ES2440651T3 - Method to diagnose preeclampsia - Google Patents

Abstract

A method for diagnosing, in a pregnant person, pre-eclampsia or the propensity to develop it, said method comprising measuring the level of the sFlt-1 polypeptide in a sample of a subject, where dichamuestra is serum or plasma and comparing the level of sFlt-1 with the level of sFlt-1 in a reference sample where an increase in the level of the sFlt-1 polypeptide in relation to said reference is a diagnosis of depreeclampsia or a propensity to develop preeclampsia.

Description

Method to diagnose preeclampsia

Field of the Invention

In general, this invention relates to the detection and treatment of subjects who have preeclampsia or eclampsia.

Background of the invention

Preeclampsia is a syndrome of hypertension, edema and proteinuria that affects 5-10% of pregnancies and results in substantial fetal and maternal morbidity and mortality. Preeclampsia is responsible for at least 200,000 maternal deaths worldwide per year. The symptoms of preeclampsia typically appear after the twentieth week of pregnancy and are usually detected by a routine check of the mother's blood pressure and urine. However, these control methods are ineffective in diagnosing the syndrome at an early stage, which could reduce the risk of the subject or the developing fetus, if effective treatment is available.

There is currently no known cure for preeclampsia. Preeclampsia can vary in severity from mild to life threatening. A mild form of preeclampsia can be treated with bed rest and frequent control. For moderate to severe cases, hospitalization is recommended and a medication is prescribed to treat blood pressure or anticonvulsant medications to prevent attacks. If the condition becomes life-threatening for the mother or the baby, the pregnancy is interrupted and the baby is born prematurely.

The proper development of the fetus and placenta is mediated by several growth factors. One of these growth factors is the vascular endothelial growth factor (VEGF). VEGF is a specific mitogen of endothelial cells, an angiogenic inducer and a mediator of vascular permeability. It has also been shown that VEGF is important for the repair of glomerular capillaries. VEGF binds as a homodimer to one of two transmembrane homolog tyrosine kinase receptors, the fms type tyrosine kinase receptor (Flt-1) and the kinase domain receptor (KDR), which are differentially expressed in endothelial cells obtained from Many different tissues. The Flt-1 but not the KDR, has a high expression in trophoblast cells that contribute to the formation of the placenta. Placental growth factor (P1GF) is a member of the VEGF family that is also involved in the development of the placenta. P1GF is expressed by cytotrophoblasts and syncytiotrophoblasts and is capable of inducing proliferation, migration and activation of endothelial cells. P1GF binds as a homodimer to the Flt-1 receptor, but not to the KDR receptor. Both P1GF and VEGF contribute to mitogenic activity and angiogenesis that are critical for the development of the placenta.

A soluble form of the Flt-1 receptor (sFlt-1) was recently identified in a culture medium of human umbilical vein endothelial cells and subsequently in vivo expression in placental tissue was demonstrated. The sFlt-1 is a variant of Flt-1 receptor support that lacks the transmembrane and cytoplasmic domains. SFlt-1 binds to VEGF with high affinity, but does not stimulate the mitogenesis of endothelial cells. It is believed that sFlt-1 acts as a "physiological fossa" to negatively regulate the VEGF signaling pathway. Therefore, the regulation of the sFlt-1 levels acts by modulating the VEGF and the VEGF signaling pathways. Careful regulation of the signaling pathways of VEGF and P1GF is critical to maintain proper proliferation, migration and angiogenesis through trophoblast cells in the developing placenta. There is a need for methods to accurately diagnose subjects at risk of suffering from or having preeclampsia, particularly before the onset of the most severe symptoms. A treatment is also needed.

Vuorela and colleagues reveal that preeclampsia is associated with high levels of sVEGFR, that is, sFlt-1 in the amniotic fluid during the third trimester ("Amniotic Fluid-Soluble Vascular Endothelial Growth Factor Receptor-1 in Preeclampsia", OBSTETRICS AND GYNECOLOGY, NEW YORK, NY, USA, vol. 95, no 3., March 2000 (2000-03) PAGES 353-357).

Zhou and collaborators reveal that cytotrophoblasts present in the placenta of women with a high degree of preeclampsia release approximately twice the amount of sFlt-1 compared to controls (“Vascular endothelial growth factor ligands and receptors that regulate human cytotrophoblast survival are dysregulated in severe preeclampsia and hemolysis, elevated liver enzymes, and low platelets syndrome ”, AM J PATHOL, vol. 160,

No. 4, April 2002 (2002-04), pages 1405-1423).

Polliotti and collaborators reveal that maternal P1GF serum during the second trimester is lower in patients with preeclampsia than in controls (“Second-trimester maternal serum placental growth factor and vascular endothelial growth factor for predicting severe, early-onset preeclampsia”, OBSTETRICS AND GYNECOLOGY , NEW YORK, NY, USA, vol. 101, No. 6, June 2003 (2003-06), pages 1266-1274).

Taylor and colleagues reveal that P1GF-free serum levels decrease in patients with preeclampsia as soon as they reach 15-19 weeks of gestation (“Longitudinal serum concentrations of placental growth

factor: Evidence for abormal placental angiogenesis in pathologic pregnancies ”, AMERICAN JOURNAL OF OBSTETRICS & GYNECOLOGY, MOSBY, SAN LUIS, MO, USA UU., Vol. 188, No. 1, January 1, 2003 (2003-0101), pages 177-182).

Compendium of the Invention

A means to diagnose and effectively treat preeclampsia and eclampsia has been discovered before symptoms occur.

Using gene expression analysis, we have found that sFlt-1 levels are markedly elevated in placental tissue samples from pregnant women suffering from preeclampsia. It is known that sFlt-1 antagonizes VEGF and P1GF by acting as a "physiological fossa" and, in preeclamptic or eclantic women, sFlt-1 can reduce the necessary amounts of these essential angiogenic and mitogenic factors in the placenta. An excess of sFlt-1 can also cause eclampsia by breaking down the endothelial cells that maintain the blood brain barrier and / or the endothelial cells that line the choroid plexus of the brain, thus leading to cerebral edema and the attacks observed in eclampsia. Compounds that increase VEGF and P1GF levels are administered to a subject to treat or prevent preeclampsia or eclampsia by counteracting the effects of the elevated sFlt-1 level. In addition, antibodies directed against sFlt1 are used to competitively inhibit the binding of VEGF or P1GF to this sFlt-1, thereby increasing free VEGF and P1GF levels. Also antisense nucleobases oligomers and RNA interference are used to reduce sFlt-1 levels. Finally, the present invention provides the use and control of sFlt-1, VEGF and P1GF as detection tools for a diagnosis and early treatment of preeclampsia or eclampsia, or a predisposition to it.

A method for treating or preventing preeclampsia or eclampsia in a subject is described by administering to the subject a compound capable of binding to sFlt-1, where administration is performed over a period of time and in an amount sufficient to treat or prevent preeclampsia or eclampsia in a subject.

In a related aspect, a method for treating or preventing preeclampsia or eclampsia in a subject is described by administering to the subject a compound (e.g. nicotine, theophylline, adenosine, nifedipine, minoxidil or magnesium sulfate) that increases the level of a growth factor capable of binding to sFlt-1, where administration is performed for a period of time and in an amount sufficient to treat or prevent preeclampsia or eclampsia in a subject.

In another related aspect, the invention provides a method for treating or preventing preeclampsia or eclampsia in a subject by administering to the subject a purified sFlt-1 antibody or an antigen binding fragment thereof for a period of time and in an amount sufficient to treat or prevent preeclampsia or eclampsia in a subject.

In another related aspect, a method for treating or preventing preeclampsia or eclampsia in a subject is described, administering to the subject an antisense nucleobase oligomer complementary to at least a portion of a sFlt-1 nucleic acid sequence being sufficient administration to treat or prevent preeclampsia or eclampsia in a subject. In one embodiment, the antisense nucleobase oligomer has a length of 8 to 30 nucleotides.

In another related aspect, a method for treating or preventing preeclampsia or eclampsia in a subject is described. The method involves the step of administering to a subject a double helix RNA (RNAds) containing at least a portion of a sFlt-1 nucleic acid sequence, the administration being sufficient to treat or prevent preeclampsia or eclampsia in the subject. . In one embodiment, double helix RNA is processed in small interference RNA (siRNA) 19 to 25 nucleotides in length.

In various embodiments of the foregoing aspects, the candidate compound is a growth factor such as vascular endothelial growth factor (VEGF), including all isoforms such as VEGF189, VEGF121

or VEGF165; placental growth factor (P1GF) including all isoforms; or fragments of these. In preferred embodiments, the candidate compound is an antibody that binds to sFlt-1. In other embodiments of the above aspects, the method also involves administering to an individual an antihypertensive compound. In still other embodiments of the above aspects, the subject is a pregnant woman, a woman after childbirth or a non-human animal (for example, a cow, a horse, a sheep, a pig, a goat, a dog or a cat ).

In another aspect, a method for treating or preventing preeclampsia or eclampsia is described. The method involves administering to an individual in need of such treatment an effective amount of a pharmaceutical composition comprising a VEGF or P1GF polypeptide. In one embodiment, the composition contains a VEGF polypeptide. In another embodiment, the composition contains a P1GF polypeptide.

In a related aspect, a method for treating or preventing preeclampsia or eclampsia is described. This method involves the administration to a subject in need of said treatment of an effective amount of a composition.

pharmaceutical comprising a nucleic acid molecule encoding VEGF or P1GF. In one embodiment, the composition contains a VEGF nucleic acid molecule. In another embodiment, the composition contains a P1GF nucleic acid molecule.

In another related aspect, a method for treating or preventing preeclampsia or eclampsia in a subject is described. The method involves the step of administering to the subject a compound (eg, chemical compound, polypeptide, peptide, antibody or a fragment thereof) that inhibits the binding of the growth factor to an sFlt-1 polypeptide, the administration being sufficient to treat or prevent preeclampsia or eclampsia in a subject. In one embodiment, the compound binds to sFlt-1 and blocks the growth factor binding.

In various embodiments of the foregoing aspects, the method also includes the step of administering an antihypertensive compound to a subject (eg, adenosine, nifedipine, minoxidil and magnesium sulfate). In another of the above aspects, the subject is a pregnant woman, a woman after childbirth or a non-human animal (for example, a cow, a horse, a sheep, a pig, a goat, a dog or a cat).

Specifically, in a first aspect of the invention, there is provided a method for diagnosing in a pregnant person preeclampsia or the propensity to develop it, said method comprising measuring the level of sFlt-1 polypeptide in a sample of a person, where said sample is a serum or a plasma, and comparing the level of sFlt-1 with the level of sFlt-1 in a reference sample where an increase in the level of sFlt-1 polypeptide with respect to this reference sample is an indicator diagnosis of preeclampsia or a propensity to develop it.

In a related aspect, the invention provides a method for diagnosing a subject with preeclampsia or eclampsia or a propensity to develop them, determining the levels of sFlt-1, VEGF and P1GF polypeptide in a serum or plasma sample of a subject and calculating the relationship between the levels of sFlt-1, VEGF or P1GF using a metric [sFlt-1 / P1GF / VEGF] where an increase in the subject's sample with respect to the diagnostic reference preeclampsia or eclampsia in this person. In this embodiment, the metric is a preeclampsia antiangiogenic index (PAAI): [sFlt-1 / VEGF + P1GF], where the PAAI is used as an indicator of angiogenic activity. In one embodiment, a PAAI value greater than 20 indicates preeclampsia or eclampsia. In another embodiment, the levels of sFlt-1, VEGF or P1GF polypeptide are determined by an immunological assay, such as ELISA.

In another aspect of the invention, there is provided a method for diagnosing preeclampsia in a pregnant person or a propensity to develop it, said method comprising measuring the level of free P1GF polypeptide in a serum or plasma sample of said subject where said subject is studying its first trimester of pregnancy and where a statistically significant decrease in free P1GF is an early indicator of the existence of preeclampsia.

In a further aspect of the invention, there is provided a method for diagnosing preeclampsia in a pregnant person, said method comprising measuring the free P1GF level and creatinine in a person's urine sample, where a decrease in the P1GF ratio as Creatinine in the sample when compared to a control is an indicator for the diagnosis of preeclampsia.

In several embodiments of the above aspects, the sample is serum, plasma or urine. In one embodiment a level of sFlt-1 greater than 2 ng / ml is an indicator of preeclampsia or eclampsia.

In preferred embodiments of the above aspects, the level of sFlt-1 polypeptide measured is the level of free, bound or total sFlt-1 polypeptide.

In other preferred embodiments of the above aspects, the level of VEGF or P1GF is the level of free VEGF or P1GF.

In another aspect, the invention provides a method for diagnosing in a subject the condition or propensity to develop preeclampsia or eclampsia. This method involves measuring the level of a sFlt-1 nucleic acid molecule in a serum or plasma sample of the subject and comparing it with a reference sample, diagnosing an alteration in the levels of preeclampsia or eclampsia in the subject. , or diagnosing a propensity to develop preeclampsia or eclampsia.

In another aspect, a method for diagnosing in a subject the condition or propensity to develop preeclampsia or eclampsia is described. This method involves determining the nucleic acid sequence of a sFlt-1, VEGF or P1GF gene in a subject and comparing it with a reference sequence, diagnosing in the subject an alteration in the subject's nucleic acid sequence. which changes the level of the gene product in the subject a state of preeclampsia or eclampsia, or a propensity to develop preeclampsia or eclampsia. In one embodiment, the alteration is a polymorphism in the nucleic acid sequence.

In various embodiments of the above aspects, the sample of a body fluid is urine, serum or plasma of the subject in which sFlt-1, VEGR or P1GF can normally be detected. The subject is a pregnant person. In other embodiments of the above aspects, at least one of the levels measured is the level of sFlt-1 (free, bound

or total). In other embodiments of the above aspects, when the level of VEGF is measured then the level of sFlt-1 or P1GF is also measured. In various embodiments of the above aspects, an increase in the level of sFlt-1 nucleic acid or polypeptide with respect to a reference is a diagnostic indicator of preeclampsia or eclampsia. In other embodiments of the above aspects, a reduction in the level of free VEGF polypeptide or VEGF nucleic acid with respect to a reference is a diagnostic indicator of preeclampsia or eclampsia. In other embodiments of the above aspects, a reduction in the level of free P1GF polypeptide or P1GF nucleic acid with respect to a reference is a diagnostic indicator of preeclampsia or eclampsia.

In additional embodiments of the above aspects, levels are measured twice or more and a change in levels between measurements is a diagnostic indicator of preeclampsia or eclampsia. In a preferred embodiment, the level of sFlt-1 increases from the first measurement to the next measurement. In another preferred embodiment, the level of VEGF or P1GF decreases from the first measurement to the next measurement.

A diagnostic kit for the diagnosis of preeclampsia or eclampsia is also described in a subject comprising a nucleic acid sequence, or a fragment thereof, selected from the group consisting of a nucleic acid molecule of sFlt-1, VEGF and P1GF , or a sequence complementary to this, or any combination of these. In a preferred embodiment, the kit comprises at least two probes for the detection of a sFlt-1, VEGF or P1GF nucleic acid molecule.

A kit for the diagnosis of preeclampsia or eclampsia is also described in a subject comprising a means to detect an sFlt-1, VEGF or P1GF polypeptide, and any combination thereof. In one embodiment, the detection medium is selected from the group consisting of an immunological assay, an enzymatic assay and a colorimetric assay. In other embodiments of the above aspects, the kit diagnoses a propensity to develop preeclampsia or eclampsia in a pregnant or non-pregnant woman. In preferred embodiments of the above aspects, the kit detects sFlt-1 or P1GF. In other preferred embodiments of the above aspects, when the kit detects VEGF, then sFlt-1 or P1GF is also detected.

In another aspect, a method for identifying a compound that improves preeclampsia or eclampsia is described, including the method of contacting a cell that expresses a sFlt-1, VEGF or P1GF nucleic acid molecule with a candidate compound, and comparing the level of expression of the nucleic acid molecule in the cell that has come into contact with the candidate compound with the level of expression in a control cell that has not come into contact with the candidate compound, identifying an alteration in the expression of the sFlt-1, VEGF or P1GF nucleic acid molecule that the candidate compound is a compound that improves preeclampsia or eclampsia.

In one embodiment, the alteration is a reduction in the level of sFlt-1. In another embodiment, the alteration is an increase in the level of VEGF or P1GF. In other embodiments, the alteration is in transcription or translation. In another embodiment, when the method identifies a candidate compound that increases the expression of VEGF, the candidate compound also increases the expression of P1GF or reduces the expression of sFlt-1.

In another aspect, a pharmaceutical composition is described that includes a VEGF or P1GF polypeptide or a portion thereof, formulated in a pharmaceutically acceptable carrier.

In a related aspect, a pharmaceutical composition comprising a P1GF nucleic acid molecule, or a portion thereof, formulated in a pharmaceutically acceptable carrier is described. In one embodiment, the composition also contains a VEGF nucleic acid molecule, or a portion thereof.

In another aspect, a composition is described comprising a purified antibody or an antigen binding fragment thereof that specifically binds to sFlt-1. In a preferred embodiment, the antibody prevents the binding of a growth factor to sFlt-1. In another embodiment, the antibody is a monoclonal antibody. In other preferred embodiments, the antibody or an antigen binding fragment thereof is a human or humanized antibody. In other embodiments, the antibody lacks an Fc portion. In yet other embodiments, the antibody is an F (ab ') 2 structure, a Fab or an Fv structure. In other embodiments, the antibody or antigen binding fragment thereof is present in a pharmaceutically acceptable carrier.

In another aspect, a method for identifying a compound that improves preeclampsia or eclampsia is described. This method includes contacting a cell expressing an sFlt-1, VEGF or P1GF polypeptide with a candidate compound and comparing the level of expression of the polypeptide in the cell that has been in contact with the candidate compound with the level of expression of the polypeptide. in a control cell that has not been in contact with the candidate compound, identifying an alteration in the expression of the sFlt-1 VEG or P1GF polypeptide that the candidate compound is a compound that improves preeclampsia or eclampsia. In one embodiment, the alteration in expression is tested using an immunological assay, an enzymatic assay or an immunoassay. In one embodiment, the alteration in expression is a reduction in the level of sFlt-1. In another embodiment, the alteration in expression is an increase in the level of VEGF or P1GF.

In another aspect, a method for identifying a compound that improves preeclampsia or eclampsia is described. The method includes contacting a cell expressing an sFlt-1, VEGF, or P1GF polypeptide with a candidate compound, and comparing the biological activity of the polypeptide in the cell that has been in contact with the candidate compound with the level of biological activity. in a control cell that has not been in contact with the candidate compound, identifying an increase in the biological activity of the sFlt-1, VEGF or P1GF polypeptide that the candidate compound is a compound that improves preeclampsia or eclampsia. In one embodiment, the increase in biological activity is tested using an immunological assay, an enzymatic assay or an immunoassay. In one embodiment, the alteration in expression is a reduction in the activity of sFlt-1. In another embodiment, the alteration in expression is an increase in the activity of VEGF or P1GF.

In another aspect, a method for identifying a compound that improves preeclampsia or eclampsia is described. The method involves the detection of the binding between an sFlt-1 polypeptide and a growth factor in the presence of a candidate compound, identifying a reduction in binding with respect to the binding between the sFlt-1 polypeptide and the absent growth factor of the candidate compound that the candidate compound is a compound that improves preeclampsia or eclampsia. In one embodiment, the growth factor is VEGF. In another embodiment, the growth factor is P1GF.

In another aspect, a method for identifying a polypeptide or a fragment thereof is described, which prevents binding between an sFlt-1 polypeptide and a growth factor. The method involves the detection of the binding between an sFlt-1 polypeptide and a growth factor in the presence of the candidate polypeptide, identifying a reduction in binding, with respect to the binding between the sFlt-1 polypeptide and the growth factor in the absence. of the candidate polypeptide, that the candidate polypeptide is a polypeptide that prevents binding between an sFlt-1 polypeptide and a growth factor. In one embodiment, the growth factor is VEGF. In another embodiment, the growth factor is P1GF.

In another aspect, a method for identifying a compound that improves preeclampsia or eclampsia is described, which comprises detecting the binding of an sFlt-1 polypeptide and a candidate compound, where a compound that binds to the sFlt-1 polypeptide improves preeclampsia or eclampsia.

In a related aspect, a compound identified according to the previous aspect is described, where the compound is a polypeptide that specifically binds to an sFlt-1 polypeptide and prevents the binding of the sFlt-1 polypeptide to VEGF or P1GF. In a preferred embodiment, the polypeptide is an antibody. In another preferred embodiment, the polypeptide is a fragment of sFlt-1, VEGF or P1GF.

In preferred embodiments of the above aspects, the compound that improves preeclampsia or eclampsia reduces the expression levels or biological activity of sFlt-1. In preferred embodiments of the above aspects, the compound that improves preeclampsia or eclampsia increases the expression levels or biological activity of VEGF or P1GF.

For the purposes of the present invention, the following abbreviations and terms are defined below.

By "alteration" is meant a change (increase or decrease) in the expression levels of a gene or a polypeptide as detected by conventional methods known in the art such as those described above. As used herein, an increase or reduction includes a 10% change in expression levels, preferably a 25% change, more preferably a 40% change and even more preferably a 50% or greater change in the levels. Expression levels "Alteration" may also indicate a change (increase or decrease) in the biological activity of any of the polypeptides of the invention (for example, sFlt-1, VEGF or P1GF). Examples of biological activity of P1GF or VEGF include receptor binding as measured by immunoassays, ligand binding assays or Scatchard graph analysis, and induction of cell proliferation or migration as measured by BrdU labeling, experiments. cell count or quantitative assays for DNA synthesis such as the incorporation of 3H-thymidine. Examples of biological activity for sFlt-1 include binding to P1GF and VEGF measured by immunoassays, ligand binding assays or Scatchard graph analysis. This document describes other examples of biological activity for each of the polypeptides. As used herein, an increase or reduction includes a 10% change in biological activity, preferably a 25% change, more preferably a 40% change and even more preferably a 50% or greater change in activity. biological

By "antisense nucleobase oligomer" is meant a nucleobase oligomer, regardless of length, which is complementary to the coding strand or mRNA of an sFlt-1 gene. By "nucleobase oligomer" is meant a compound that includes a chain of at least eight nucleobases, preferably at least twelve and even more preferable at least sixteen bases, linked together by linking groups. This definition includes both natural and unnatural oligonucleotides, both modified and unmodified, as well as oligonucleotide mimetics such as protein nucleic acids, blocked nucleic acids and arabinonucleic acids. In the nucleobase oligomers of the invention, numerous nucleobases and linking groups can be employed, including those described in US Pat. UU. 20030114412 and 20030114407. The nucleobase oligomer can also be directed to translation start and stop sites. Preferably, the oligomer

Antisense nucleobases comprise about 8 to 30 nucleotides. The antisense nucleobase oligomer may also contain at least 40, 60, 85, 120 or more consecutive nucleotides that are complementary to the sFlt-1 mRNA or DNA, and may be as long as the mRNA or the full length gene.

By "compound" is meant any small molecule chemical compound, antibody, nucleic acid molecule or polypeptide, or fragments thereof.

By "chimeric antibody" is meant a polypeptide comprising at least the antigen-binding portion of an antibody molecule bound to at least part of another protein (typically a constant immunoglobulin domain).

By "double stranded RNA (cRNA)" is meant a molecule of ribonucleic acid composed of both a sense chain and an antisense chain. CRNAs are typically used to mediate RNA interference.

By "expression" is meant the detection of a gene or polypeptide by conventional methods known in the art. For example, expression of a polypeptide is often detected by Western blotting, DNA expression is often detected by Southern blotting or polymerase chain reaction (PCR), and RNA expression is often detected by Northern blotting. , PCR or RNAse protection assays.

By "fragment" is meant a portion of a molecule of polypeptide or nucleic acid. This portion preferably contains at least 10%, 20%, 30%, 40%, 50% or 60% of the entire length of the nucleic acid molecule or reference polypeptide. A fragment may contain 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 nucleotides or amino acids.

By "homologue" is meant any gene or protein sequence that carries at least 30% homology, more preferably 40%, 50%, 60%, 70%, 80%, and even more preferably 90 homology %

or greater with a known gene or protein sequence throughout the length of the comparison sequence. A "homologous" protein can also have at least one biological activity of the comparison protein. In the case of polypeptides, the length of the comparison sequences will generally be at least 16 amino acids, preferably at least 20 amino acids, more preferably at least 25 amino acids and even more preferably 35 amino acids or more. In the case of nucleic acids, the length of the comparison sequences will generally be at least 50 nucleotides, preferably at least 60 nucleotides, more preferably at least 75 nucleotides and even more preferably at least 110 nucleotides. "Homology" may also refer to a substantial analogy between an epitope used to generate antibodies and the protein or fragment thereof to which the antibodies are directed. In this case, homology refers to a similarity sufficient to induce the production of antibodies that can specifically recognize the protein in question.

By "humanized antibody" is meant a variant of an immunoglobulin amino acid sequence or fragment thereof that is capable of binding to a predetermined antigen. Usually the antibody will contain both the light chain and at least the variable domain of a heavy chain. The antibody may also include the CH1, hinge, CH2, CH3 or CH4 regions of the heavy chain. The humanized antibody comprises a flanking region (FR) that has substantially the amino acid sequence of a human immunoglobulin and a complementarity determining region (CDR) that has substantially the amino acid sequence of a non-human immunoglobulin (the "import" sequences) .

Generally, a humanized antibody has one or more amino acid residues introduced from a source that is not human. In general, the humanized antibody will comprise substantially all of at least one, and typically two variable domains (Fab, Fab ', F (ab') 2, Fabc, Fv) in which all or substantially all CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a consensus sequence of human immunoglobulin. The optimally humanized antibody will comprise at least a portion of a constant region of immunoglobulin (Fc), typically that of a human immunoglobulin.

By "complementarity determining region (CDR)" are understood the three hypervariable sequences in the variable regions within each of the light and heavy immunoglobulin chains.

By "flanking region (FR)" are understood the amino acid sequences located on either side of the three hypervariable sequences (CDR) of the light and heavy immunoglobulin chains.

The FR and CDR regions of the humanized antibody do not need to correspond precisely to the parental sequences, for example, the import CDR or the consensus FR can be mutagenized by substitution, insertion or removal of at least one residue, so that the rest of CDR or FR at that site does not correspond to the consensus or import antibody. However, such mutations will not be extensive. Normally, at least 75%, preferably 90% and even more preferably at least 95% of the antibody residues

Humanized will correspond to those of the parental FR and CDR sequences.

By "hybridizing" is meant to form base pairs with a double stranded molecule between complementary polynucleotide sequences, or portions thereof, under various stringent conditions. (See, for example, Wahl and Berger (1987) Methods Enzymol. 152: 399; Kimmel, Methods Enzymol. 152: 507, 1987). For example, the concentration of rigorous salt will normally be less than about 750 mM NaCl and 75 mM trisodium citrate, preferably less than about 500 mM NaCl and 50 mM trisodium citrate and even more preferably less than about 250 mM NaCl and trisodium citrate. mM. A low stringency hybridization can be obtained in the absence of an organic solvent, for example, formamide, while a high stringency hybridization can be obtained in the presence of at least 35% formamide, and more preferably at least about 50% formamide. Rigorous temperature conditions will usually include temperatures of at least about 30 ° C, more preferably of at least about 37 ° C and even more preferably of at least about 42 ° C. Additional variable parameters, such as hybridization time, detergent concentration, for example, sodium dodecyl sulfate (SDS) and inclusion or exclusion of transporter DNA, are well known to those skilled in the art. Various levels of rigor are achieved by combining these various conditions as necessary. In a preferred embodiment, hybridization will be performed at 30 ° C in 750 mM NaCl, 75 mM trisodium citrate and 1% SDS. In a more preferred embodiment, hybridization will be performed at 37 ° C in 500 mM NaCl, 50 mM trisodium citrate, 1% SDS, 35% formamide and denatured salmon sperm DNA (DNAs) at 100 μg / ml. In a more preferred embodiment, hybridization will be performed at 42 ° C in 250 mM NaCl, 25 mM trisodium citrate, 1% SDS, 50% formamide and DNAs at 200 µg / ml. Useful variations of these conditions will be apparent to those skilled in the art.

For most applications, the washing steps that follow the hybridization will also vary in rigor. Washing stringency conditions can be defined by salt concentration and temperature. As indicated above, washing rigor can be increased by reducing the salt concentration or increasing the temperature. For example, a concentration of rigorous salt for the washing steps will preferably be less than about 30 mM NaCl and 3 mM trisodium citrate, and even more preferably less than about 15 mM NaCl and 1.5 mM trisodium citrate. The stringent temperature conditions for the washing steps will normally include a temperature of at least about 25 ° C, more preferably of at least about 42 ° C and even more preferably of at least about 68 ° C. In a preferred embodiment, the washing steps will be performed at 25 ° C in 30 mM NaCl, 3 mM trisodium citrate and 0.1% SDS. In a more preferred embodiment, the washing steps will be performed at 42 ° C in 15 mM NaCl, 1.5 mM trisodium citrate and 0.1% SDS. In a more preferred embodiment, the washing steps will be performed at 68 ° C in 15 mM NaCl, 1.5 mM trisodium citrate and 0.1% SDS. Other variations of these conditions will be apparent to those skilled in the art. Hybridization techniques are well known to those skilled in the art and are described, for example, in Benton and Davis (Science 196: 180, 1977); Grunstein and Hogness (Proc. Natl. Acad. Sci., United States 72: 3961, 1975); Ausubel et al. (Current Protocols in Molecular Biology, Wiley Interscience, New York, 2001); Berger and Kimmel (Guide to Molecular Cloning Techniques, 1987, Academic Press, New York); and Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York.

"Intrauterine growth retardation (IUGR)" means a syndrome that results in a birth weight that is less than 10 percent of the expected fetal weight for the gestational age of the fetus. The current criteria of the World Health Organization for a low birth weight is a weight less than 2500 g (5 lbs, 8 oz) or below the 10 percent percentile for gestational age according to the tables of United States birth weight for gestational age by race, parity, and sex of the child (Zhang and Bowes, Obstet. Gynecol. 86: 200-208, 1995). These babies with low birth weight are also called "small for gestational age (SGA)". Preeclampsia is a condition that is known to be associated with IURG or SGA.

"Measurement system" means a measurement. A measurement system can be used, for example, to compare the levels of a polypeptide or nucleic acid molecule of interest. Examples of suitable measurement systems include, but are not limited to, mathematical formulas or algorithms, such as relationships. The measurement system to be used is the one that best discriminates between the levels of sFlt-1, VEGF or P1GF in a subject who has preeclampsia or eclampsia and a normal control subject. Depending on the measurement system used, the diagnostic indicator of eclampsia or preeclampsia may be significantly above or below a reference value (for example, of a control subject who does not have preeclampsia or eclampsia).

The level of sFlt-1 is measured by measuring the amount of free, bound (ie, bound to the growth factor) or total (bound + free) sFlt-1. VEGF or P1GF levels are determined by measuring the amount of free P1GF or free VEGF (ie, not bound to sFlt-1). An illustrative measurement system is [sFlt-1 / (VEGF + P1GF)], also called preeclampsia antiangiogenic index (PAAI).

The term "anti-angiogenesis index of preeclampsia (PAAI)" means the ratio of sFlt-1 / VEGF + P1GF used as an indicator of antiangiogenic activity. A PAAI greater than 20 is considered indicative of preeclampsia or with

risk of preeclampsia.

By "operably linked" is meant that a gene and one or more regulatory sequences are connected in such a way that gene expression is allowed when the appropriate molecules (eg transcription activating proteins) bind to the regulatory sequences.

By "pharmaceutically acceptable carrier" is meant a vehicle that is physiologically acceptable to the treated animal while maintaining the therapeutic properties of the compound with which it is administered. An exemplary pharmaceutically acceptable carrier is saline physiological solution. Other physiologically acceptable carriers and their formulations are known to the person skilled in the art and are described, for example, in Remington's Pharmaceutical Sciences, (20th edition), A. Gennaro, editor, 2000, Lippincott, Williams & Wilkins, Philadelphia, Pennsylvania, United States.

By "placental growth factor (P1GF)" is meant a mammalian growth factor that is homologous to the protein defined by the GenBank accession number P49763 and that has biological activity of P1GF. P1GF is a glycosylated homodimer that belongs to the VEGF family and can be found in two different isoforms through alternative splicing mechanisms. P1GF is expressed by cyto- and syncytiotrophoblasts of the placenta and the biological activities of P1GF include induction of proliferation, migration and activation of endothelial cells, particularly trophoblast cells.

By "preeclampsia" is the multisystemic disorder characterized by hypertension with proteinuria or edema, or both, glomerular dysfunction, cerebral edema, hepatic edema or coagulation abnormalities due to pregnancy or the influence of a recent pregnancy. Preeclampsia usually occurs after the twentieth week of gestation. Preeclampsia is generally defined as some combination of the following symptoms: (1) a systolic blood pressure (PS)> 140 mmHg and a diastolic PS> 90 mmHg after 20 weeks of gestation (usually measured twice, separated by a period 4-168 hours),

(2) fresh-start proteinuria (1 + per test strips in urinalysis,> 300 mg of urine protein collected for 24 hours, or a single random urine sample having a protein / creatinine ratio> 0.3), and (3) resolution of hypertension and proteinuria at 12 weeks after delivery. Severe preeclampsia is usually defined as (1) a diastolic PS> 110 mmHg (usually measured twice, separated by a period of 4168 hours) or (2) proteinuria characterized by a measure of 3.5 g or more in urine protein 24-hour collection or two random urine samples with a protein level of at least 3+ measured by test strips. In preeclampsia, hypertension and proteinuria usually appear within a seven day interval. In severe preeclampsia, severe hypertension, severe proteinuria and HELLP syndrome (hemolysis, elevated liver enzymes and low platelet levels) or eclampsia may appear simultaneously or only one symptom at a time. Occasionally, severe preeclampsia can lead to the development of seizures. This severe form of the syndrome is called "eclampsia." Eclampsia may also include dysfunction or lesions in various organs or tissues such as the liver (for example, hepatocellular injury, periportal necrosis) and the central nervous system (for example, cerebral edema and cerebral hemorrhage). It is believed that the etiology of seizures is secondary to the development of cerebral edema and focal spasm of small blood vessels in the kidney.

By "protein" or "polypeptide" or "polypeptide fragment" is meant any chain of more than two amino acids, regardless of post-translational modification (eg, glycosylation or phosphorylation), which constitutes all or part of a natural polypeptide or peptide, or which constitutes an unnatural polypeptide or peptide.

By "reduced or inhibited" is meant the ability to produce a total reduction preferably of 20% or more, more preferably of 50% or more and even more preferably of 75% or more of the level of protein or nucleic acid, detected by the Assays mentioned above (see "expression") compared to samples not treated with antisense nucleobase oligomers or dsRNA used for RNA interference.

"Small interference RNA (siRNA)" means an isolated rRNA molecule, preferably with a length greater than 10 nucleotides, more preferably with a length greater than 15 nucleotides and even more preferably with a length greater than 19 nucleotides that is used. to identify the target gene or mRNA to be degraded A range of 19-25 nucleotides is the most preferred size for siRNAs. siRNAs can also include short hairpin RNAs that include the two strands of an siRNA duplex within a single RNA molecule. The siRNA includes any form of rRNA (proteolytically cleaved products of a larger rRNA, partially purified RNA, essentially pure RNA, synthetic RNA and recombinantly produced RNA) as well as altered RNA that differs from natural RNA by the addition, removal, substitution and / or alteration of one or more nucleotides Such alterations may include the addition of a materi to the non-nucleotide, such as the ends of the RNA from 21 to 23 nt or internally (in one or more nucleotides of the RNA). In a preferred embodiment, the RNA molecules contain a 3 'hydroxyl group. Nucleotides in the RNA molecules of the present invention may also comprise unconventional nucleotides, including unnatural nucleotides or deoxyribonucleotides. Collectively, all these altered RNAs are called RNA analogs. The siRNAs of the present invention need only be sufficiently similar to natural RNA to have the ability to mediate RNA interference (RNAi). As used herein, RNAi refers to

targeted ATP-dependent cleavage and degradation of a specific mRNA molecule through the introduction of small interfering RNA or cRNA into a cell or organism. As used herein, "mediated RNAi" refers to the ability to distinguish or identify the RNA to be degraded.

By "soluble Flt-1 (sFlt-1)" (also known as sVEGF-R1) is the soluble form of the Flt-1 receptor, which is homologous to the protein defined by the GenBank accession number U01134, and which has biological activity of sFlt-1. The biological activity of an sFlt-1 polypeptide can be tested using any conventional method, for example, by testing the binding of sFlt-1 to VEGF. SFlt-1 lacks the transmembrane domain and the cytoplasmic tyrosine kinase domain of the Flt-1 receptor. SFlt-1 can bind VEGF and P1GF binding with high affinity, but cannot induce proliferation or angiogenesis and is therefore functionally different from Flt-1 and KDR receptors. SFlt-1 was initially purified from human umbilical endothelial cells and subsequently demonstrated to be produced by trophoblast cells in vivo. As used herein, the sFlt-1 includes any member or isoform of the sFlt-1 family.

By "specifically bound" is meant a compound or antibody that recognizes and binds to a polypeptide of the invention, but that does not substantially recognize or bind to other molecules in a sample, for example, a biological sample, which includes so a natural polypeptide of the invention. In one example, an antibody that specifically binds to sFlt-1 does not bind to Flt-1.

By "subject" is meant a mammal, including, but not limited to, a human being or a non-human mammal such as a cow, horse, dog, sheep or cat. This definition includes pregnant, postpartum and non-pregnant mammals.

By "substantially identical" is meant an amino acid sequence that differs only by conservative amino acid substitutions, for example, substitution of one amino acid for another of the same class (eg, glycine for valine, lysine for arginine, etc.) or for one or more non-conservative substitutions, deletions or insertions located at positions in the amino acid sequence that do not destroy the function of the protein. Preferably, the amino acid sequence has a homology of at least 70%, more preferably of at least about 80% and even more preferably of at least about 90%, with another amino acid sequence. The methods for determining identity are available in computer programs available to the public. Computer program methods for determining the identity between two sequences include, but are not limited to, the GCG program package (Devereux et al., Nucleic Acids Research 12: 387, 1984), BLASTP, BLASTN and FASTA (Altschul et al., J. Mol. Biol. 215: 403 (1990) The well-known Smith Waterman algorithm can also be used to determine identity.The BLAST program is available to the public at NCBI and other sources (BLAST Manual, Altschul et al., NCBI NLM NIH , Bethesda, Maryland 20894, United States; BLAST 2.0 at http://www.ncbi.nlm.nih.gov/blast/) These software programs compare similar sequences by assigning degrees of homology to various substitutions, deletions and other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine, valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lisi na, arginine; and phenylalanine, tyrosine.

By "symptoms of preeclampsia" means any of the following: (1) a systolic blood pressure (PS)> 140 mmHg and a diastolic PS> 90 mmHg after 20 weeks of gestation, (2) new proteinuria (1)

+ per urinalysis test strip,> 300 mg of urine protein collected for 24 hours, or a random urine protein / creatinine ratio> 0.3) and (3) resolution of hypertension and proteinuria at 12 weeks after delivery . Symptoms of preeclampsia may also include renal dysfunction and endotheliosis or glomerular hypertrophy. "Symptoms of eclampsia" means the development of any of the following symptoms due to pregnancy or the influence of a recent pregnancy: seizures, coma, thrombocytopenia, hepatic edema, pulmonary edema and cerebral edema.

By "therapeutic amount" is meant an amount that when administered to a patient suffering from preeclampsia or eclampsia is sufficient to cause a qualitative or quantitative reduction in the symptoms of preeclampsia or eclampsia as described herein. A "therapeutic amount" can also mean an amount that when administered to a patient suffering from preeclampsia or eclampsia is sufficient to produce a reduction in the expression levels of sFlt-1 or an increase in the expression levels of VEGF or P1GF as It is measured in the tests described in this document.

By "treatment" is meant the administration of a compound or a pharmaceutical composition for prophylactic and / or therapeutic purposes. To "treat a condition" or use for "therapeutic treatment" refers to the administration of a treatment to a subject already suffering from a condition to improve the condition of the subject. Preferably, the subject has been diagnosed with preeclampsia or eclampsia based on the identification of any of the characteristic symptoms described below or through the use of the diagnostic methods described herein. To "prevent the condition" refers to a prophylactic treatment of a subject who is not yet sick, but who is prone or at risk of developing a particular condition. Preferably, it is determined that a subject is at risk of developing preeclampsia or eclampsia using the

diagnostic methods described in this document. Thus, in the claims and embodiments, the treatment is administration to a mammal for therapeutic or prophylactic purposes.

By "trophoblast" is meant the layer of mesoectodermal cells that cover the blastocyst that erodes the uterine mucosa and through which the embryo receives nutrition from the mother; The cells contribute to the formation of the placenta.

By "vascular endothelial growth factor (VEGF)" is meant a mammalian growth factor that is homologous to the growth factor defined in US Pat. UU. 5,332,671; 5,240,848; 5,194,596; and Charnock-Jones et al. (Biol. Reproduction, 48: 1120-1128, 1993) and has biological activity of VEGF. VEGF exists as a glycosylated homodimer and includes at least four differently spliced isoforms. The biological activity of native VEGF includes the promotion of selective growth of vascular endothelial cells or umbilical vein endothelial cells and induction of angiogenesis. As used herein, the VEGF includes any member or isoform of the VEGF family (for example VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, VEGF189, VEGF165, or VEGF 121) . Preferably, the VEGF is the VEGF121 or VEGF165 isoform (Tischer et al., J. Biol. Chem. 266, 11947-11954, 1991; Neufed et al. Cancer Metastasis 15: 153-158, 1996), which is described in the patents of USA UU. 6,447,768; 5,219,739; and 5,194,596. Also included are mutant forms of VEGF such as VEGF with KDR selectivity and VEGF with Flt selectivity described in Gille et al. (J. Biol. Chem. 276: 3222-3230, 2001). Although human VEGF is preferred, the invention is not limited to human forms and may include other animal forms of VEGF (eg, mouse, dog or chicken).

By "vector" is meant a DNA molecule, normally derived from a plasmid or bacteriophage, into which DNA fragments can be inserted or cloned. A recombinant vector will contain one or more unique restriction sites, and may be able to replicate autonomously in a defined host or vehicle organism such that the cloned sequence is reproducible. A vector contains a promoter operably linked to a gene or coding region such that, after transfection in a recipient cell, an RNA is expressed.

Other features and advantages of the invention will be apparent upon the following description of the preferred embodiments, and of the claims.

Brief Description of the Drawings

Figure 1 shows mRNA and protein expression of sFlt-1 in preeclampsia. Figure 1A shows the expression of placental sFlt-1 mRNA from three patients with preeclampsia (P1, P2, P3) and three full-term pregnancies with normal pressure (N1, N2, N3) as determined by Northern blot analysis. The major band (7.5 kb) is the full-length Flt-1 mRNA and the smallest and most abundant band (3.4 kb) is the alternatively bound sFlt-1 mRNA. GAPDH is included as a control and the arrowhead indicates 28S RNA. Patients P1 and P2 had severe preeclampsia while patient P3 had mild preeclampsia. Figure 1B is a graph showing the levels of sFlt-1 in the serum of patients with mild preeclampsia (mild PE), patients with severe preeclampsia (severe PE), and full-term (normal) normotensive pregnant women. The sFlt-1 levels were measured by an ELISA performed for sFlt-1 using a commercially available kit (R&D Systems, Minneapolis, United States). As additional controls, patients with deliveries before term were included for other reasons (before reaching term) to rule out specific changes in gestational age. The number of patients tested is shown in parentheses on the X axis. Samples were collected before delivery (t = 0) and 48 hours after delivery (t = 48). Figure 1C is a graph showing the ratios of antiangiogenesis indices (PAAI = sFlt-1 / (VEGF + P1GF) at the time of delivery (t = 0), determined by ELISA for all patients described in Figure 1B.

Figure 2A-2F are photomicrographs showing the antiangiogenic effect of excess sFlt-1 in preeclampsia. Endothelial tube trials were performed using serum from four normal pregnant control women and four patients with preeclampsia. A representative experiment of a normal control and a patient with preeclampsia are shown. Figures 2A, 2B and 2C show tests performed using serum from a normal patient, while Figures 2D, 2E and 2F show tests performed using serum from a patient with preeclampsia. In Figure 2A, t = 0, (10% serum of a normal pregnant woman at term); in Figure 2B, t = 48 (10% serum of a normal pregnant woman 48 hours after delivery); in Figure 2 C, t = 0 + sFlt-1 exogenous (10 ng / ml); in Figure 2D, t = 0 (10% serum of a pre-eclamptic woman before delivery); in Figure 2E, t = 48 (10% serum of a pre-eclamptic woman 48 hours after delivery), and in Figure 2F, t = 0 + exogenous VEGF (10 ng / ml) + P1GF (10 ng / ml) . The tube test was quantified and the average length of the +/- SEM tube is shown in pixels at the bottom of each panel.

Figures 3A and 3B are graphs showing that inhibition of VEGF and P1GF induced vasodilation of renal microvessels by sFlt-1. Figure 3A demonstrates that the increase in relaxation responses of rat renal arterioles to sFlt-1 (S), VEGF (V), P1GF (P) was measured with three different doses. V + and P + represent vasodilatory responses of individual reagents in the presence of sFlt-1 at 100 ng / ml. All experiments are

performed on 6 different dissected rat renal microvessels and the data is shown as mean +/- SEM. The * represents the statistical significance with p <0.01 compared to the individual reagents alone. Figure 3B shows the increase in physiological dose relaxation responses: VEGF 100 pg / ml (V), P1GF 500 pg / ml (P), sFlt-1 10 ng / ml (S), VEGF (100 pg / ml ) + P1GF 500 pg / ml (V + P) or VEGF (100 pg / ml) + P1GF 500 pg / ml + sFlt-1 10 ng / ml (V + P + S). All experiments were performed on 6 different dissected rat renal microvessels and the data is shown as mean +/- SEM. The * represents the statistical significance with p <0.05 compared to V + P.

Figures 4A and 4B show the induction by sFlt-1 of glomerular endotheliosis. Figure 4A is a photomicrograph showing staining with hematoxylin and eosin (H&E) in a capillary occlusion in animals treated with sFlt-1 with enlarged glomeruli and swollen cytoplasm compared to controls. In animals treated with sFlt-1 after Schiff staining with periodic acid (PAS), "glomerular endotheliosis" is observed with bubble cytoplasm. All optical microscopy photographs were taken at 60 magnifications, with the original enlargement. Figure 4B is an electron micrograph of glomeruli treated with sFlt-1 that confirms the cytoplasmic inflammation of the endocapillary cells. Immunofluorescence (IF) for fibrin photographs were taken at 40 magnifications and EM photographs were taken at 2400 magnifications, original magnification. All figures were reproduced with the same magnification.

Figures 5A-5C show the sFlt-1 levels measured before and after the onset of preeclampsia by gestational age. Figure 5A is a graph showing the mean serum concentrations in pg / ml for normotensive controls (lighter line with white triangles), cases before preeclampsia (filled circles) and cases after preeclampsia - criteria samples of assessment - (filled squares) within windows 4-5 weeks of gestational age before the onset of labor. Parentheses indicate the standard error of the mean. Asterisks indicate significant differences with respect to control samples within the same gestational age window after logarithmic transformation: * p <0.05, ** p <0.01, *** p <0.001. Figure 5B is a graph showing the mean serum concentrations of sFlt-1 in pg / ml for cases before and after the onset of preeclampsia at intervals of weeks before preeclampsia. PE indicates the arithmetic mean of 43 samples of assessment criteria (obtained during or after the onset of preeclampsia). The average gestational age (days) is indicated in parentheses below each time interval. The horizontal line indicates the level in the samples of the assessment criteria. The vertical lines mark the period of � 5 weeks before preeclampsia. Figure 5C is a graph showing the mean serum concentrations of sFlt-1 in pg / ml per gestational age window for normotensive controls and cases before preeclampsia, after excluding samples obtained during the subsequent 5 weeks at the beginning of preeclampsia. There are no significant differences.

Figures 6A-6C show P1GF levels before and after preeclampsia by gestational age. Figure 6A is a graph showing the levels of P1GF in all samples obtained before labor and delivery. Parentheses indicate the standard error of the mean. The asterisks indicate significant differences with respect to the control samples within the same interval after the logarithmic transformation: ** p <0.01, *** p <0.001. Figure 6B is a graph showing the mean serum concentrations of P1GF in pg / ml for cases before and after the onset of preeclampsia within intervals of weeks before preeclampsia. PE indicates the arithmetic mean of 43 samples of assessment criteria (obtained during or after the onset of preeclampsia). The average gestational age (days) is indicated in brackets below each time interval. The horizontal line indicates the level in the samples of the assessment criteria. The vertical lines mark the period of � 5 weeks before preeclampsia. Figure 6C is a graph showing the mean serum concentrations of P1GF in pg / ml per gestational age window for normotensive controls and cases of onset of preeclampsia.

Figures 7A and 7B show the levels of sFlt-1 and P1GF by state and severity of preeclampsia. Figure 7A is a graph showing the arithmetic mean serum concentrations of sFlt-1 (black bars) and P1GF (white bars) at 23-32 weeks gestation in controls and cases (before the onset of clinical condition ) with mild preeclampsia, severe preeclampsia, preeclampsia with onset <37 weeks, preeclampsia with a small baby for gestational age (SGA), and preeclampsia with onset <34 weeks. Sample numbers are recorded below each pair of columns. The adjustment for gestational age and body mass index resulted in minor changes without affecting the level of meaning. Figure 7B is a graph showing the arithmetic mean serum concentrations of sFlt-1 (black bars) and P1GF (white bars) at 33-41 weeks gestation in controls and cases (before the onset of clinical condition) with mild preeclampsia, severe preeclampsia, preeclampsia with onset <37 weeks and preeclampsia with an SGA baby. Sample numbers are recorded below each pair of columns. The adjustment for gestational age and body mass index resulted in minor changes without affecting the level of meaning.

Detailed description

It has been found that sFlt-1 levels are elevated in blood serum samples taken from preeclamptic women. sFlt-1 binds to VEGF and P1GF with high affinity and blocks the mitogenic and angiogenic activity of

These growth factors. Thus, sFlt-1 is an excellent diagnostic marker for preeclampsia and VEGF and P1GF can be used to treat preeclampsia. In addition, therapeutic agents have been discovered that interfere with the binding of purified sFlt-1 to VEGF or P1GF, or agents that increase biologically active levels of VEGF or P1GF, and that can be used to treat or prevent preeclampsia or eclampsia in a subject. . Such agents include, but are not limited to, antibodies against sFlt-1, oligonucleotides for antisense or RNAi that reduce sFlt-1 levels, compounds that increase levels of VEGF or P1GF, and small molecules that bind sFlt-1 and block the growth factor binding site. The invention also characterizes methods for measuring the levels of growth factors; The methods can be used as diagnostic tools for early detection of preeclampsia or an increased risk of developing preeclampsia or eclampsia.

Although the detailed description presented in this document specifically refers to sFlt-1, VEGF or P1GF it will be apparent to a specialist in the field that the detailed description can also be applied to sFlt-1, VEGF or members of the P1GF family, isoforms and / or variants, and growth factors that have been shown to bind sFlt-1. The following examples are to illustrate the invention and should not be construed as limiting.

Example 1. Increase in mRNA Levels of sFlt-1 and Protein in Pregnant Women with Preeclampsia

In an attempt to identify new secreted factors that play a pathological role in preeclampsia, a gene expression profile of placental tissue of women with and without preeclampsia has been performed using Affymetrix U95A microarray chips. It has been discovered that the gene for sFlt-1 was positively regulated in women with preeclampsia.

To confirm the positive regulation of sFlt-1 in preeclampsia, Northern blots have been made to analyze mRNA levels of placental sFlt-1 (Figure 1A) and ELISA assays to measure serum protein levels of sFlt-1 (Figure 1B) in preeclamptic pregnant women compared to normotensive pregnant women. Preeclampsia was defined as (1) a systolic blood pressure (PS)> 140 mmHg and a diastolic PS> 90 mmHg after 20 weeks of gestation, (2) new-onset proteinuria (1 + by urinalysis test strips,> 300 mg protein in urine collected for 24 hours, or a random urine protein / creatinine ratio> 0.3), and (3) resolution of hypertension and proteinuria at twelve weeks after delivery. Patients with hypertension, proteinuria or underlying renal disease were excluded. The patients were divided into mild and severe preeclampsia based on the presence or absence of nephritic interval proteinuria (> 3 g of urine protein collected for 24 hours or a protein / creatinine ratio in urine greater than 3.0). The mean urine / protein creatinine ratios in the mild preeclampsia group were 0.94 +/- 0.2 and in the severe preeclampsia group they were 7.8 +/- 2.1. The average gestational ages of the various groups were as follows: normal 38.8 +/- 0.2 weeks, mild preeclampsia 34 +/- 1.2 weeks, severe preeclampsia 31.3 +/- 0.6 weeks, and preterm 29.5 +/- 2.0 weeks. Placental samples were obtained immediately after delivery. Four random samples were taken from each placenta, placed in RNA stabilization solution (Ambion, Austin, Texas, United States) and stored at -70 ° C. RNA isolation was performed using a Qiagen RNAeasy Maxi kit (Qiagen, Valencia, California, United States).

An increase in both placental sFlt-1 mRNA and maternal serum sFlt-1 protein was detected in preeclamptic pregnant women compared to normotensive pregnant women. The mean serum level of sFlt-1 was almost four times higher in patients with severe preeclampsia compared to normal control pregnant women. To exclude the possibility that this effect was due to the previous gestational age of pre-eclamptic cases, sFlt-1 levels were also measured in normotensive women with similar gestational age and with premature delivery for other reasons (gestational ages of 23-36 weeks) and no significant differences were found in this group compared to normal normotensive pregnancies. The probes used for Northern blots were obtained by PCR and included a 500 bp fragment in the human Flt-1 cDNA coding region of pUC118, and a GAPDH cDNA that was used as a normalization control.

In a normal pregnancy there is a balance between the pro and antiangiogenic factors secreted by the placenta that is necessary for proper placental development. It was hypothesized that in preeclampsia, the increase in the production of sFlt-1 and the reduction in the production of VEGF and P1GF shifts the balance in favor or against angiogenesis. To treat net antiangiogenic activity, serum levels of VEGF and P1GF were measured and serum levels of P1GF and VEGF were found to be lower in patients with preeclampsia compared to normal control patients (mean P1GF, 235.3 + / - 45.3 pg / ml versus 464 +/- 116.6 pg / ml) as described (Tidwell et al., Am. J. Obstet. Gynecol., 184: 1267-1272, 2001). When sFlt-1 was incorporated, the levels of VEGF and P1GF in an antiangiogenic index, or PAAI, as an indicator of net antiangiogenic activity, it was discovered that pre-eclamptic patients could be clearly separated from normal patients and that the PAAI It seemed to correlate with the severity of preeclampsia (Figure 1C). This PAAI can be used as a diagnostic tool for the detection of preeclampsia in pregnant women.

Example 2. The Serum of Women with Preeclampsia Inhibits Angiogenesis in an In Endothelial Tube Test

Vitro

It was hypothesized that the excess of circulating sFlt-1 in patients with preeclampsia causes endothelial dysfunction and leads to an antiangiogenic state. To solve this, an endothelial tube assay was used as an in vitro model of angiogenesis. Matrigel reduced growth factor (7 mg / ml, Collaborative Biomedical Products, Bedford, Massachusetts, United States) was placed in wells (100 ml / well) of a previously cooled 48-well cell culture plate and incubated at 37 ° C for 25-30 minutes to allow polymerization. Human umbilical vein endothelial cells (30,000 + 300 ml of serum-free endothelial basal medium, Clonetics, Walkersville, Maryland, United States) were treated in passes 3-5 with 10% patient serum, placed in the coated wells with Matrigel and incubated at 37 ° C for 12-16 hours. The tube formation was then evaluated by means of a 4x inverted phase contrast microscope (Nikon Corporation, Tokyo, Japan) and quantitatively analyzed (tube area and total length) using the simple PCI image analysis software.

The conditions of the tube formation assay were adjusted such that normal human umbilical vein endothelial cells only formed tubes in the presence of exogenous growth factors such as VEGF. Under these conditions, it was discovered that although the serum of normotensive women induced endothelial cells to form symmetric tube-like structures, the serum of women with preeclampsia inhibited the formation of the tube (Figure 2). Notably, at 48 hours after delivery this antiangiogenic effect had disappeared, suggesting that the inhibition of the tubes detected with the serum of patients with preeclampsia was probably due to a circulating factor released by the placenta. When sFlt-1 was added to normotensive serum in doses similar to those found in patients with preeclampsia, tube formation did not occur, mimicking the effects observed with the serum of preeclamptic women. When exogenous VEGF and P1GF were added to the assay using pre-eclamptic serum, tube formation was restored (Figure 2). For these assays recombinant human VEGF, human P1GF and human Flt-1Fc were used. These results suggested that the antiangiogenic properties of preeclamptic serum were due to antagonism of VEGF and P1GF by endogenous sFlt-1. These results also suggested that the addition of purified VEGF and / or P1GF can reverse or mitigate the preeclamptic state and can be used therapeutically.

Example 3. sFlt-1 Inhibits Vasodilatation Induced by VEGF and P1GF from Kidney Microvessels

The causative role of sFlt-1 in vasoconstriction was determined using an in vitro microvascular reactivity experiment. Microvascular reactivity experiments were performed as previously described using rat renal microvessels (Sato et al., J. Surg. Res., 90: 138-143, 2000). Microvessels of renal arteries (70-170 μm internal diameter) were dissected from rat kidneys using a 10-magnification dissection microscope at 60 magnifications (Olympus Optical, Tokyo, Japan). The microvessels were placed in an isolated microvessage chamber, cannulated with double glass micropipettes measuring 30-60 μm in diameter and fixed with a 10-0 nylon mono-filament suture (Ethicon, Somerville, New Jersey) . Oxygenated Krebs buffer solution (95% oxygen and 5% carbon dioxide) heated at 37 ° C was continuously circulated through the vessel chamber and a reservoir containing a total of 100 ml of the solution. The vessels were pressurized at 40 mmHg in a flowless state using a burette gauge filled with Krebs buffer. With an inverted microscope (40 x 200 x; Olympus CK2, Olympus Optical) connected to a camcorder, the image of the vessel was projected onto a black and white television monitor. An electronic dimension analyzer (Living System Instrumentation, Burlington, Vermont, United States) was used to measure the internal diameter of the lumen. The measurements were recorded with a recorder with printed chart (Graphtec, Irvine, California, United States). The vessels were allowed to bathe in the microvessage chamber for at least 30 minutes before any intervention. In all experimental groups, the relaxation responses of the renal microvessels were examined after the pre-contraction of the microvessels with U46619 (thromboxane agonist) at 40-60% of their initial diameter at a distension pressure of 40 mmHg. Once the steady state tone was reached, responses to various reagents such as VEGF, P1GF and sFlt-1 were examined. For these assays recombinant rat VEGF, mouse P1GF and mouse Flt-1Fc were used. All drugs were applied extraluminally. Measurements were made when the response had stabilized (usually 2-3 minutes after administering the drug). In each vessel, one to four interventions were performed. The vessels were washed with a Krebs buffer solution and allowed to equilibrate in a Krebs buffer solution without drug for 20-30 minutes between the interventions.

It was found that sFlt-1 alone did not produce significant vasoconstriction, however it blocked the increase in dose response in vasodilation induced by VEGF or P1GF (Figure 3A). In addition, it was found that VEGF and P1GF, at the physiological levels observed in pregnancy, induced significant dose-dependent arteriolar relaxation, and that this effect was blocked by the addition of 10 ng / ml of sFlt-1, an observed concentration in women with severe preeclampsia (Figure 3B). This result suggested that circulating sFlt-1 in patients with preeclampsia may oppose vasorelaxation, thus contributing to hypertension. These results confirm the conclusion that sFlt-1 is responsible for many of the clinical and pathological symptoms of preeclampsia, including hypertension. Inhibition of sFlt-1, through the use of targeted antibodies, for example, could reverse the effects of protein in preeclamptic women and such sFlt-1 inhibitors could

potentially used as therapeutic agents.

Example 4. Effects of sFlt-1 in an Animal Model of Preeclampsia

Based on the previous results, it was hypothesized that the addition of exogenous sFlt-1 would produce hypertension and proteinuria in an animal model. Adenoviruses expressing sFlt-1 have been shown to produce sustained systemic levels of sFlt-1 associated with significant antitumor activity (Kuo et al., Proc. Natl. Acad. Sci. United States, 98: 4605-4610, 2001). This recombinant adenovirus encoding murine sFlt-1 was injected into the tail vein of pregnant Sprague-Dawley rats on day 8-9 of pregnancy. As controls, adenoviruses encoding murine Fc and sFlk1-Fc (fusion protein of the Flk1 electrodomain of mouse VEGF receptor 1 and Fc protein) were used in equivalent doses. Flk1 has been shown to bind VEGF, but not P1GF. Therefore, sFlk-1Fc was chosen as a control to help distinguish between the anti-VEGF activity and the anti-P1GF activity of sFlt-1.

1 x 109 pfu of Ad Fc, Ad sFlt-1, or Ad sFlk-1Fc were injected into pregnant and non-pregnant Sprague-Dawley rats by means of injections into the tail vein. These adenoviruses have been previously described (Kuo et al., Supra) and were generated at the Harvard Vector Core Laboratory. In pregnant rats, adenoviruses were injected on day 8-9 of pregnancy (in the first part of the second trimester) and blood pressure was measured on day 16-17 of pregnancy (in the first part of the third trimester). In non-pregnant animals, PS was measured on day 8 after adenovirus injection. PS were measured in rats after anesthesia with sodium pentobarbital (60 mg / kg, i.p.). The carotid artery was isolated and cannulated with a 3-Fr high fidelity micropuntant catheter connected to a pressure transducer (Millar Instruments, Houston, Texas, United States). The Millar Mikro-Tip catheter was advanced inside the artery to record blood pressure. Blood pressure and heart rate were recorded on a recorder with printed chart (model 56-1X 40-006158, Gould Instrument Systems, Cleveland, Ohio, United States) and an average of a 10-minute period was calculated. Blood, tissue and urine samples were obtained before sacrificing the rats. Urine albumin was measured by a conventional test strip and quantified by a competitive enzyme linked immunoassay (ELISA) as described elsewhere in this document (Cohen et al., Kidney Intl., 45: 1673-1679, 1994). Urine creatinine was measured by a colorimetric procedure kit of picric acid (Sigma, St. Louis, Missouri, United States). Intra-arterial blood pressures were measured in the first part of the third trimester of pregnancy to mimic the natural pathology of preeclampsia. These experiments were also performed on non-pregnant female Sprague-Dawley rats to determine whether the effects of sFlt-1 are direct or indirect through their effects on the placenta. It was confirmed by Western blot analysis that the systemic levels of sFlt-1 on the day of blood pressure measurement were within the range of 25-350 ng / ml in the various animals treated with sFlt-1 on the day of PS measurement. Table 1 shows blood pressure and proteinuria in the different experimental groups.

Table 1. Blood pressure and proteinuria in rats

N

MAP (mmHg) U alb ratio: cr

Fc (P)

5 75.6 ± 11.1 62 ± 21

sFlt-1 (P)

4 109.0 ± 19.3 * 6923 ± 658 *

sFlk-1Fc (P)

4 72.8 ± 14.7 50 ± 32

Fc (NP)

5 89.3 ± 5.7 138 ± 78

sFlt-1 (NP)

6 117.9 ± 129 * 12947 ± 2776 *

sFlk-1Fc (NP)

4 137.3 ± 2.3 * 2269 ± 669 *

Pregnant (P) and non-pregnant (NP) rats were given adenoviruses expressing Fc (control), sFlt-1 or sFlk-1Fc protein. Mean arterial blood pressure (MAP = diastolic + 1/3 pulse pressure in mmHg) ± SEM and albumin ratio: Cr in urine (mg of albumin per gram of creatinine) ± SEM was measured eight days later, corresponding to the First part of the third trimester in pregnant rats. N = the number of animals in each experimental group. The * represents statistical significance with p <0.01 compared to the control group (Fc).

Pregnant rats treated with sFlt-1 had significant hypertension and a significant nephrotic interval albuminuria compared to Fc controls. Nonpregnant rats given sFlt-1 also developed hypertension and proteinuria. Notably, non-pregnant rats treated with sFlk-Fc developed hypertension and proteinuria, while pregnant rats treated with sFlk-Fc did not. Therefore, in pregnancy, VEGF antagonism is only sufficient to produce preeclampsia, possibly due to the presence of high levels of P1GF. In the non-pregnant state, in which P1GF is practically absent, VEGF antagonism is only sufficient to break the pro / antiangiogenic balance and produce renal pathologies similar to those associated with preeclampsia. Various staining techniques were used to examine the renal lesion observed in all rats treated with sFlt-1 (Figure 4). Kidneys collected from the rats in Bouin solution were fixed, cut into sections and stained with H&E and PAS dyes. For electron microscopy, the renal tissue was fixed in glutaraldehyde, included in a mixture of araldite-epon and renal sections were cut

ultrafine (1 µm), stained with toluene blue and evaluated using Zeiss EM 10 with various magnifications. Immunofluorescence for fibrin deposits within the glomeruli was performed using polyclonal antifibrin antibody (ICN, Switzerland). Global and diffuse glomerular endotheliosis was the renal lesion universally observed in rats treated with sFlt-1. Glomerular augmentation was detected with occlusion of the capillary loops due to swelling and hypertrophy of the endocapillary cells. Numerous apparent protein resorption droplets were observed in glomerular epithelial cells. No segmental glomerulosclerosis was observed. Isolated "double contours" and a focal deposition of fibrin within the glomeruli were seen. This finding of fibrin deposition in the absence of a significant mesangial interposition is similar to that described as typical of the preparatory phase of the human condition (Kincaid-Smith, Am. J. Kidney Dis., 17: 144-148 , 1991). Immunofluorescence for fibrin showed foci of fibrin deposition within the glomeruli of animals treated with sFlt-1, but not of animals treated with Fc. Non-pregnant rats treated with sFlk1 developed the same lesion. In fact, when sFlk1 was used with the same levels as sFlt-1, renal injury was more severe in non-pregnant rats, since there are fewer circulating proangiogenic molecules to antagonize sFlt-1. These results suggested that elevated sFlt-1 levels may be responsible for glomerular endotheliosis associated with preeclampsia, but that this effect was independent of the placenta, since glomerular changes were detected in non-pregnant rats as well as in pregnant rats. These results also suggested that the antagonism of VEGF and P1GF is important in the pathology of preeclampsia since hypertension and proteinuria occurred in non-pregnant mice treated with sFlk-1 but not in pregnant mice treated with sFlk-1 where levels of P1GF are high.

The animal model created in this document can be used as an experimental model to test new therapeutic compounds. Using this animal model, the efficacy of potential therapeutic compounds as well as pharmacology and toxicity can be studied.

Example 5. Effects of sFlt-1 in an Animal Model of Eclampsia

Exogenous sFlt-1 is injected into pregnant rats in the first part of their second trimester of pregnancy. The rats are then monitored and tested during the first part of their third trimester to detect the development of eclampsia. Assays used for the detection of eclampsia may include MRI of rat brains for the development of edema, EEG of the rat brain for seizure development and histology of rat brains to determine if endothelial injury has occurred. along the blood brain barrier and the choroid plexus using specific endothelial markers.

The animal model created in this document can be used as an experimental model to test new therapeutic compounds. Using this animal model, the efficacy of potential therapeutic compounds such as pharmacology and toxicity can be studied.

Example 6: The P1GF / Creatinine Ratio in Urine Serves as a Diagnosis of Preeclampsia

Urine samples were obtained from 10 women at 16 weeks gestation (five normal, four with mild preeclampsia and one with severe preeclampsia). These samples were provided by Dr. Ravi Thadhani of the Massachusetts General Hospital. The mean ratios of free P1GF / urine creatinine (pg of P1GF per mg of creatinine) for normal pregnant women were 78 +/- 10.7 and for the four women with mild preeclampsia were 33 +/- 5.0 and for the patient with severe preeclampsia it was 17. Thus, an alteration in the relationship between P1GF and urine creatinine is useful as a diagnostic indicator of preeclampsia in a patient.

Example 7: Protein levels sFlt-1 and P1GF as a Diagnostic Indicator of Preeclampsia and Eclampsia in women.

For this study, archived samples of the calcium assay for the prevention of preeclampsia were used to analyze the gestational models of sFlt-1, free P1GF and free VEGF circulating in normotensive and preeclamptic pregnancies. The calcium trial for the prevention of preeclampsia, or CPEP, was a randomized double-blind clinical trial conducted during 1992-1995 to assess the effects of the daily supplement of 2 g of elemental calcium or placebo on the incidence and severity of preeclampsia ( Levine et al., N. Engl. J. Med. 377: 69-76, 1997; Levine et al., Control Clin. Trials 17: 442-469, 1996). Healthy nulliparous women with simple pregnancies with between 13 and 21 weeks of gestation in 5 participating US medical centers were included and followed up to 24 hours after delivery using a common protocol and identical data collection forms. At the time of inclusion, all CPEP participants had a blood pressure <135/85 mmHg, and none had renal dysfunction or proteinuria. Gestational age was determined by ultrasound examination. Serum samples were obtained from participants before inclusion in the trial (13-21 weeks), at 2629 weeks, at 36 weeks if they were still pregnant, and when hypertension or proteinuria was detected. The "samples of assessment criteria" were samples obtained during or after the onset of symptoms and signs of preeclampsia, but before labor and delivery as described elsewhere in this document (Levine et al., 1996 , supra). Blood samples obtained from the CPEP test are

obtained thanks to the collaboration of Dr. Richard Levine in the NIH.

Participants

Subjects with complete information on the results were selected, from which serum samples had been obtained at <22 weeks and who had a male who was born alive. Of the 4589 CPEP participants, 253 who lost follow-up were excluded, 21 whose pregnancy ended before 20 weeks, 13 absences of maternal or perinatal outcomes, 4 without a history of smoking, 9 with hypertension not verified by the groups for review, and another 32 with children born dead, leaving 4257 women with adequate information and live births. Among these, 2156 had male babies. After excluding a woman whose baby had a chromosomal abnormality, 381 with gestational hypertension and 43 without an initial serum sample, 1731 women remained. Of these, 175 developed preeclampsia and 1556 remained normotensive throughout the pregnancy.

As the calcium supplement had no effect on the risk and severity of preeclampsia and was not related to the concentrations of pro and antiangiogenic molecules, cases and controls were chosen without taking into account the treatment in the CPEP. For each case of preeclampsia, a normotensive control was selected, equivalent in all inclusion sites in terms of gestational age in the collection of the first serum sample (before one week) and the freezer storage time at -70 ° C ( within 12 months) 120 equivalent pairs (“cases” and “controls”) were randomly chosen for the analysis of the 657 serum samples obtained before delivery (Table 2, presented below). The average gestational age in the collection of the first serum sample was 112.8 and 113.6 days in the cases and controls respectively; The average duration of storage in the freezer was 9.35 and 9.39 years.

TABLE 2: Characteristics of cases and controls in the inclusion in CPEP and its newborn babies

features

Cases (n = 120) Controls (n = 120)

Age (years)

20.8 ± 4.5 20.2 ± 3.46

Height (cm)

161.0 ± 6.7 163.0 ± 6.9

Weight (kg)

71.0 ± 19.4 66.8 ± 17.1

Body mass index

27.3 ± 6.8 25.1 ± 6.1 **

Systolic blood pressure (mmHg)

109.5 ± 8.8 105.7 ± 9.0 **

Diastolic blood pressure (mmHg)

62.0 ± 7.9 59.4 ± 7.4 **

Previous pregnancy loss [n (%)]

23 (19.2) 25 (20.8)

Current smoker [n (%)]

9 (7.5) 13 (10.8)

Private social insurance [n (%)]

8 (6.7) 13 (10.8)

Married [n (%)]

25 (20.8) 24 (20.0)

Race / ethnicity

White, not Hispanic [n (%)]

24 (20.0) 35 (29.2)

Hispanic White [n (%)]

21 (17.5) 14 (11.7)

African American [n (%)]

69 (57.5) 68 (56.7)

Other, unknown [n (%)]

6 (5.0) 3 (2.5)

Birth weight (g)

3100 ± 796 3255 ± 595

Delivery <37 weeks [n (%)]

29 (24.2) 9 (7.5) **

Small for gestational age (<tenth percentile) [n (%)]

18 (15.0) 4 (3.3) **

Mean ± standard deviation unless indicated * p <0.05 ** p <0.01

For this study, hypertension was defined as a diastolic blood pressure of at least 90 mmHg on two separate occasions for a period of 4-168 hours. Severe hypertension was defined as a diastolic blood pressure of at least 110 mmHg on two separate occasions for a period of 4-168 hours, or one occasion if the woman had received antihypertensive therapy. Proteinuria was defined as 300 mg or more of protein in a 24-hour urine collection, two random urine samples separated by a period of 4-168 hours containing at least 1+ protein by means of a test strip, a single urine sample with a protein / creatinine ratio of at least 0.35, or a single random urine sample containing at least 2+ protein per test strip. Severe proteinuria was diagnosed by a 24-hour urine collection sample containing at least 3.5 g of protein or by two random urine samples with at least 3+ protein by means of a test strip. Preeclampsia was defined as hypertension and proteinuria that occurred with a difference of less than 7 days; severe preeclampsia was defined as preeclampsia with severe hypertension, severe proteinuria, HELLP syndrome (hemolysis, elevated liver enzymes and low platelet concentration) or eclampsia. The onset of preeclampsia was the time of detection of the first elevation of blood pressure or proteinuria in

the urine sample that led to the diagnosis of preeclampsia.

Small size for gestational age (SGA) was defined as a birth weight less than the 10% percentile for gestational age according to the United States birth weight tables for gestational age by race, parity and sex of the baby (Zhang and Bowes 1995, supra).

Procedures

The trials were conducted at the Beth Israel Deaconess Medical Center by laboratory personnel who were unaware of the diagnosis of the patients and other relevant clinical information. Samples were randomly ordered for analysis. Bound enzyme immunosorbent assays (ELISA) for human sFlt-1, free P1GF and free VEGF were performed according to the manufacturer's instructions, using kits purchased from R&D Systems (Minneapolis, Minneapolis, United States). Aliquots of serum samples that had been stored at -70 ° C were thawed at room temperature, diluted with BSA / Tris buffered saline and incubated for 2 hours in a 96-well plate previously coated with capture antibody directed against sFlt- 1, P1GF or VEGF. The wells were then washed three times, incubated 20 minutes with a substrate solution containing hydrogen peroxide and tetramethylbenzidine and the reaction was quenched with 2N sulfuric acid. The optical density was determined at 450 nm (wavelength correction at 550 nm). All trials were performed in duplicate. Protein concentrations were calculated using a standard curve derived from known concentrations of the respective recombinant proteins. If the difference between the duplicates exceeded 25%, the test was repeated and the initial results were discarded. The trials had sensitivities of 5, 7 and 5 pg / ml for sFlt-1, P1GF and VEGF, respectively, with coefficients of inter- and intra-assay variation of 7.6% and 3.3% for sFlt-1, of 11, 2% and 5.4% for P1GF, and 7.3% and 5.4% for VEGF.

Statistic analysis

In the analysis of the maternal or child characteristics to compare the categorical or continuous variables, Chi-square and t-tests were used respectively. Although the arithmetic mean values of the concentrations are provided in the text and in the figures, the statistical test was performed after a logarithmic transformation, unless otherwise indicated. The adjustment was made using logistic reversal at logarithmically transformed concentrations.

Results

Of the 120 cases, 80 developed preeclampsia and 40 developed severe preeclampsia, including 3 with HELLP syndrome and 3 with eclampsia. The patients in the case group were lower than the control patients, had a higher body mass index and had a higher initial blood pressure (Table 2). In addition, higher proportions of the patients in the case group had complicated pregnancies, either preterm birth or young children for gestational age (SGA). Case patients contributed an average of 2.9 serum samples to the study; Controls with 2.6 samples.

First, it was confirmed that in the patients with preeclampsia the levels of sFlt-1, P1GF and VEGF were altered at the time of the active condition compared to controls with a similar gestational age of this CPEP study group. The samples taken at the time of the established clinical preeclampsia (titration criteria sample) had greatly increased sFlt-1 levels, reduced P1GF levels and reduced VEGF levels compared to controls with gestational ages (4382 versus 1643 pg / ml sFlt-1, p <0.0001; 137 versus 669 pg / ml P1GF, p <0.0001; and 6.41 versus 13.86 pg / ml VEGF, p = 0.06) for cases and controls, respectively, in 23 pairs of similar gestational age) similar to previously published reports (Maynard et al., J. Clin. Invest. 111: 649-658, 2003).

To evaluate the gestational model of the levels of sFlt-1, P1GF and VEGF, the circulating concentrations of sFlt-1, P1GF and VEGF were measured from serum samples obtained from case patients and control patients within various windows of gestational age. The gestational model of the sFlt-1 protein for 120 preeclamptic women and 120 control women is shown in Figure 5A. The sFlt-1 levels in the control patients remained constant until 33-36 weeks, at which time they rose approximately 145 pg / ml per week to labor and delivery. Among patients in the cases before clinical symptoms, sFlt-1 seemed to begin to rise at 21-24 weeks, with a step elevation and a statistically significant difference from controls at 29-32 weeks (Figure 5A) . Overall, the differences between patients in cases and controls measured before the onset of clinical symptoms were 17% (p <0.05) in the middle of pregnancy. The assessment criteria samples were significantly high compared to the samples taken before the condition. To evaluate the mechanisms of the elevation of sFlt-1 before the onset of the clinical condition, we represent the concentrations of sFlt-1 in all preeclamptic women for weeks before the onset of preeclampsia (Figure 5B). The mean concentrations of sFlt-1 in samples of case patients were represented by full weeks before the onset of preeclampsia. Starting 5 weeks before preeclampsia, sFlt-1 concentrations rose substantially up to one week before the onset of the condition, at which point they reached the concentrations observed in the

Valuation criteria samples. The increases in sFlt-1 4, 3, 2 and 1 week before preeclampsia occurred with few changes in the average gestational age and cannot be explained by an increase in the last part of the third trimester as the gestational age advanced. From 8-6 to 5 weeks before preeclampsia, the level of sFlt-1 increased to 962 pg / ml, while the average gestational age rose 31 days. Approximately one third of this increase in sFlt-1 cannot be attributed to the progress of pregnancy. When the level of sFlt-1 was represented by gestational age in controls and in cases after withdrawing the samples obtained 5 weeks before the onset of preeclampsia, no substantial differences were observed (Figure 5C). These data suggest that the highest concentration of sFlt-1 in case patients before the onset of preeclampsia is due to acute elevations in sFlt-1 in the 5 weeks prior to the onset of the clinical condition.

The gestational model of the P1GF protein was then represented in the same group of patients as shown in Figure 6A. Control P1GF protein concentrations rose during the first two trimesters, peaked at 29-32 weeks, and decreased during the last part of pregnancy. Among case patients, before preeclampsia, P1GF protein concentrations followed a similar gestational model, but were significantly lower than controls from week 13 to 16. In general, the differences in P1GF between case patients and the controls measured before the onset of clinical symptoms were 35% (p <0.0001) in the middle of pregnancy. P1GF levels in cases before the onset of preeclampsia are represented by weeks before preeclampsia (Figure 6B) and by gestational age after extracting samples <5 weeks before preeclampsia (Figure 6C). One week before the onset of preeclampsia, the concentrations approached those observed after the onset of preeclampsia (Figure 6B). Compared to controls, P1GF levels of case patients were moderately reduced since delivery, with further substantial reductions at 5 and 3 weeks before delivery. The concentrations of the control patients remained high from 17-15 to 3 weeks before delivery, and then substantially reduced. The graph showing P1GF levels excluding samples obtained 5 weeks before preeclampsia indicates a smaller reduction in cases compared to controls at 29-32 weeks of gestation and none in samples obtained from patients in cases at 33-36 weeks (Figure 6C). This suggests that the reduction in P1GF concentrations in the weeks prior to the condition was responsible for the considerably low levels of P1GF detected at the beginning of the condition (or the assessment criteria samples shown in Figure 6A).

VEGF concentrations throughout pregnancy were very low and similar in controls and in cases before preeclampsia, with the exception of a significant reduction in case patients at 3741 weeks. The average concentrations of VEGF at 23-32 weeks in cases excluding samples obtained 5 weeks before preeclampsia did not differ significantly from controls (11.6 vs. 12.8 pg / ml), while concentrations in Cases including samples 5 weeks before delivery did (5.1 vs. 12.8 pg / ml, p <0.01). At 33-41 weeks, VEGF concentrations of cases> 5 or 5 weeks before preeclampsia were higher and lower than controls respectively (11.2 pg / ml and 8.3 versus 9.7 pg / ml), although these differences were not significant.

Figure 7 represents sFlt-1 and P1GF at 23-32 weeks (Figure 7A) and 33-41 weeks (Figure 7B) by state of preeclampsia and severity. The graphs show that increases in sFlt-1 and reductions in P1GF before the onset of preeclampsia were associated with the severity of the condition, the time of onset and the presence of an SGA child. At 23-32 weeks, the levels of sFlt-1 and P1GF in case patients with an SGA child before the onset of preeclampsia were significantly higher or lower, respectively, than the corresponding concentrations in control patients with a child. GHS In addition, compared to control patients with preterm birth, patients in cases with preterm birth had a higher sFlt-1 level and a significantly lower P1GF level.

It was then determined whether circulating concentrations of P1GF and / or sFlt-1 could be used during the first trimester to identify women at risk of developing preeclampsia. Between 8-20 weeks, after adjustment for gestational age, body mass index and sFlt-1, patients with P1GF cases in the smallest quartile distribution of control values had an almost 12-fold increase in preeclampsia to <34 weeks (inequality ratio [OR] 11.7, p <0.05) compared to cases with P1GF in the upper three quartiles (Table 3). The risk of preeclampsia at <34 weeks in the lower quartile, compared to the upper quartile, increased almost 16 times (OR 15.8, p <0.01).

TABLE 3: Inequality Reasons (OR) for Early Start Preeclampsia for Control Quarters of Control P1GF at 8-20 Weeks

PE onset <34 weeks

PE onset <37 weeks

P1GF

Cases Controls OR Adj. * Cases Controls OR Adj. *

(pg / ml)

(N) (N) (95% CI) (N) (N) (95% CI)

Q4> 267.5

2 30 1.0 Reference 4 30 1.0 Reference

Q3> 128.6

one 30 0.7 (0.1-8.9) 4 30 1.3 (0.3-5.8)

267.5

219

2

Q2> 70.1

3 30 2.3 (0.3-19.3) 6 30 2.6 (0.6-12.1)

128.6

Q1 <70.1

5 30 15.8 (1.5-172.8) ** 17 30 22.3 (3.7-135.6) ***

* Inequality ratios adjusted for gestational age, body mass index, log sFlt-1 ** p <0.01 *** p <0.001 95% CI = 95% confidence limits

These results demonstrate that sFlt-1 levels begin to rise dramatically approximately 5 weeks before the onset of preeclampsia symptoms. Parallel with the increase in sFlt-1, levels of free P1GF and levels of free VEGF are reduced, suggesting that the reduction in P1GF and VEGF may be due at least partially to antagonism by sFlt-1 and not to a reduction in placental production of P1GF and VEGF. Three subgroups of preeclampsia - severe pre-eclampsia, early onset of the condition and SGA children - had higher concentrations of sFlt-1 and lower concentrations of P1GF at 23-32 weeks and at 33-41 weeks than controls or women with mild preeclampsia. There has also been a small but significant reduction in the level of P1GF free of early onset in the second trimester among women prone to develop preeclampsia. These results demonstrate that an increase in P1GF levels may be a useful agent for predicting early onset preeclampsia.

In the present invention, the gestational model of sFlt-1 in normal pregnancy is described for the first time, observing relatively stable levels throughout pregnancy followed by a constant increase beginning at 33-36 weeks. This elevation corresponds to the last gestational reduction in P1GF observed in normal pregnancy by other authors (Torry et al., J. Soc. Gynecol. Invest. 10: 178-188, 1998; Taylor et al., Am. J. Obset. Gynecol 188: 177-182, 2003) and in the results described in this document. The temporal association, together with the knowledge that sFlt-1 interferes with the ELISA measurement of P1GF (Maynard et al., Supra) suggests that the reduction of free P1GF levels during the last part of pregnancy may be due to the elevation of sFlt-1 levels. During the first and second trimester, when placental growth is needed to respond to the growing fetal demands, P1GF concentrations are high and sFlt-1 concentrations are low, creating a relatively proangiogenic state. Later in pregnancy, when it may be necessary to temper and stop placental vascular growth, there is an elevation in the antiangiogenic sFlt-1 and a resulting reduction in P1GF. In women with preeclampsia, the elevation of sFlt-1 begins earlier in pregnancy, approximately 5 weeks before the onset of symptoms, at approximately 29-32 weeks of gestation on average. Thus, in preeclampsia, antiangiogenic "brakes" can be applied too early and too strongly, resulting in an exaggeration of a normal physiological process that stops placental growth. It seems clear that the pathological placental changes that characterize preeclampsia occur early in pregnancy (10-14 weeks), before the dramatic elevation in sFlt-1. The resulting placental ischemia itself can enhance the production of sFlt-1, finally activating an increase of sFlt-1.

In addition to the large differences observed in the 5 weeks prior to the development of clinical symptoms, women destined to develop preeclampsia had small but statistically significant reductions in free P1GF levels already at 13-16 weeks gestation. This reduction in P1GF was generally not accompanied by a reciprocal increase in sFlt-1 levels. However, there was a trend of slightly higher sFlt1 levels in the cases during the first trimester although it was not statistically significant (for example, in the 17-20 week window, the average sFlt-1 levels in the cases were 865, 77 pg / ml versus 795.25 in the controls). This reduction in P1GF levels early in pregnancy could reflect a lower placental production of P1GF in pregnancies compromised by conditions such as preeclampsia or SGA. Importantly, in patients with preeclampsia complicated by SGA, a statistically significant increase was found both in the elevation of sFlt-1 and in the reduction of P1GF before the presentation of the condition. It is also possible that there is no change in the placental production of P1GF in preeclamptic patients and that the elevation of local sFlt-1 levels in the placenta may contribute to the reduction of circulating free P1GF levels. This is confirmed by the finding that placental P1GF, measured by immunohistochemistry, is not altered in preeclampsia (Zhou et al., Am J. Pathol. 160: 1405-1423. 2002).

In summary, we have shown that sFlt-1 begins to rise in preeclampsia at least 5 weeks before the onset of the clinical condition that is accompanied by a reduction in the levels of circulating free P1GF and free VEGF. The reduction of P1GF during the first trimester can be used to predict preeclampsia and the elevation of sFlt-1 can be used to predict the proximity of the clinical condition. These data together with the work in animals described above demonstrating that sFlt-1 only induces symptoms similar to those of preeclampsia in rodents suggest a probable etiological role of sFlt-1 in the pathogenesis of preeclampsia. Our limited data on SGA children and premature delivery in controls, compared with patients in the case group, suggests that the greatest alterations in protein levels observed in preeclamptic pregnancies with an SGA child are more substantial than the difference due solely to Restriction of uterine growth or premature delivery in the absence of preeclampsia.

Diagnosis

The present disclosure characterizes diagnostic tests for the detection of preeclampsia or the propensity to develop it. Free or total levels of VEGF, P1GF or sFlt-1 are measured in a sample of a subject and are used as an indicator of preeclampsia, eclampsia or the propensity to develop such conditions.

In one embodiment a measurement system is used to determine if a relationship between the levels of at least two of the proteins is indicative of preeclampsia or eclampsia. Conventional methods can be used to measure VEGF, P1GF or sFlt-1 polypeptide levels in any body fluid, including but not limited to urine, serum, plasma, saliva, amniotic fluid or cerebrospinal fluid. Such methods include immunoassay, ELISA, Western blotting using antibodies directed to VEGF, P1GF or sFlt-1 and quantitative enzyme immunoassay techniques such as those described in Ong et al. (Obstet. Gynecol. 98: 608-611, 2001 ) and Su et al. (Obstet. Gynecol., 97: 898-904, 2001). ELISAs are the preferred method of measuring levels of VEGF, P1GF or sFlt-1.

Serum levels of sFlt-1 greater than 2 ng / ml are considered a positive indicator of preeclampsia. In addition, any detectable alteration in the levels of sFlt-1, VEGF or P1GF with respect to normal levels is indicative of eclampsia, preeclampsia or the propensity to develop such conditions. Preferably sFlt-1 is measured, more preferably the measurement of VEGF and P1GF are combined with this measurement and even more preferably the 3 proteins (or mRNA levels indicative of protein levels) are measured.

In another embodiment, the PAAI (sFlt-1 / VEGF + P1GF) is used as an antiangiogenic index that is a diagnosis of preeclampsia, eclampsia or the propensity to develop such conditions. If the PAAI is over 20 then the subject is considered to have preeclampsia or has an imminent risk of developing it. The PAAI ratio (sFlt-1 / VEGF + P1GF) is simply an example of a useful measurement system that can be used as a diagnostic indicator. It is not intended to limit the invention. As a diagnostic indicator, virtually any measurement system that detects an alteration in the antiangiogenic index in the subject who has eclampsia with respect to a normal control can be used.

Expression levels of particular nucleic acids or polypeptides can be correlated with a particular disease state (for example, preeclampsia or eclampsia) and thus are useful in the diagnosis. Older oligonucleotides or fragments from a nucleic acid sequence of sFlt-1, P1GF or VEGF can be used as a probe not only to control expression, but also to identify subjects who have a genetic variation, mutation, or polymorphism in a molecule of sFlt-1, P1GF or VEGF nucleic acid that are indicative of a predisposition to develop conditions. Such polymorphisms are known to the person skilled in the art and are described by Parry et al. (Eur. J Immunogenet. 26: 321-3, 1999). Such genetic alterations may be present in the promoter sequence, an open reading frame, intronic sequence or 3'-untranslated region of an sFlt-1 gene. Information related to genetic alterations can be used to diagnose that a subject has preeclampsia, eclampsia or a propensity to develop such conditions. As indicated throughout this document, specific alterations in the levels of biological activity of sFlt-1, VEGF and / or P1GF can be correlated with the probability of preeclampsia or eclampsia, or the predisposition to these. As a result, a specialist in the field, having detected a given mutation, can test one or more systems for measuring the biological activity of the protein to determine if the mutation produces or increases the probability of preeclampsia or eclampsia.

In one embodiment, a subject who has preeclampsia, eclampsia or a propensity to develop such conditions will show an increase in the expression of a nucleic acid encoding sFlt-1 or an alteration of P1GF or VEGF levels. The methods for detecting such alterations are conventional in the art and are described in Ausubel et al., Supra. In one example, Northern blotting or real-time PCR is used to detect mRNA levels of sFlt-1, P1GF or VEGF.

In another embodiment, hybridization with PCR probes that are capable of detecting an sFlt-1 nucleic acid molecule, including genomic sequences, or closely related molecules, can be used to hybridize with a nucleic acid sequence derived from a subject having preeclampsia. or eclampsia or at risk of developing such conditions. The specificity of the probe, if it is made of a very specific region, for example, the 5 'regulatory region, or of a less specific region, for example as a conserved motive, and the astringency of hybridization or amplification (maximum, high, intermediate or low) determine whether the probe hybridizes with a natural sequence, allelic variants or other related sequences. Hybridization techniques can be used to identify mutations indicative of a preeclampsia or eclampsia in an sFlt-1 nucleic acid molecule, or they can be used to control the expression levels of a gene encoding an sFlt-1 polypeptide (for example, by analysis Northern, Ausubel et al., Supra).

In another embodiment, humans may be diagnosed with a propensity to develop preeclampsia by direct analysis of the sequence of a nucleic acid molecule of sFlt-1, VEGF or P1GF.

A subject who has preeclampsia, eclampsia or a propensity to develop such conditions will show an increase in the expression of an sFlt-1 polypeptide. An antibody that specifically binds to an sFlt-1 polypeptide can

be used to diagnose preeclampsia or eclampsia or to identify a subject at risk of developing such conditions. Various protocols for measuring an alteration in the expression of such polypeptides are known, including immunological methods (such as ELISA and RIA) and provide a basis for diagnosing preeclampsia or eclampsia or a risk of developing such conditions. Again, an increase in the level of the polypeptide is diagnostic of a subject who has preeclampsia, eclampsia or a propensity to develop such conditions.

In one embodiment, the level of sFlt-1, VEGF or P1GF polypeptide or nucleic acid or any combination of these is measured at least at two different times and an alteration in levels compared to normal reference levels over time. It is used as an indicator of preeclampsia, eclampsia or the propensity to develop such conditions.

The level of sFlt-1, VEGF or P1GF in the body fluids of a subject who has preeclampsia, eclampsia or the propensity to develop such conditions can be altered in an amount as small as 10%, 20%, 30% 40% or in an amount as large as 50%, 60%, 70%, 80% or 90% with respect to the level of sFlt-1, VEGF or P1GF in a normal control. The level of sFlt-1 present in the body fluids of a subject who has preeclampsia, eclampsia or the propensity to develop such conditions may increase 1.5 times, 2 times, 3 times, 4 times or even up to 10 times or more with respect at the levels of a normal control subject.

In one embodiment, a sample of a subject's body fluid (for example, urine, plasma, serum, amniotic fluid) is collected in the first part of pregnancy before the onset of preeclampsia symptoms. In another example, the sample may be a tissue or cell collected in the first part of pregnancy before the onset of preeclampsia symptoms. Non-limiting examples include placental tissue, placental cells, endothelial cells and leukocytes such as monocytes. In humans, for example, serum blood samples are collected from the antecubital vein of pregnant women during the first, second or third trimester of pregnancy. Preferably, the test is performed during the first trimester, for example at 4, 6, 8, 10, or 12 weeks, or during the second trimester, for example at 14, 16, 18, 20, 22 or 24 weeks. Such trials can also be performed at the end of the second trimester or at the beginning of the third trimester (approximately 28 weeks). It is preferable to measure the levels of sFlt-1, VEGF or P1GF twice during this period of time. For the diagnosis of preeclampsia or postpartum eclampsia, tests for sFlt-1, VEGF or P1GF may be performed after delivery.

In a particular example, serial blood samples may be collected during pregnancy and levels of soluble sFlt-1 can be determined by ELISA. In a study using this technique, the alternatively bound mRNA encoding sFlt-1 has high expression by trophoblast cells and the protein was easily detectable in the plasma of pregnant women. It was observed that sFlt-1 levels increased approximately 3 times between 20 and 36 weeks gestation. It was observed that the levels were significantly higher in women at high risk who subsequently developed preeclampsia (Charnock-Jones et al., J. Soc.Gynecol. Investig. 10 (2): 230, 2003).

In veterinary practice, trials may be performed at any time during pregnancy, but preferably they may be performed in the early stages of pregnancy, before the onset of preeclampsia symptoms. Since the term of pregnancies varies widely between species, the test time will be determined by a veterinarian, but will generally correspond to the test times during a human pregnancy.

The diagnostic methods described herein can be used individually or in combination with any other diagnostic method described in this document for a more accurate diagnosis of the presence, severity or estimated time of the onset of preeclampsia or eclampsia. In addition, the diagnostic methods described in this document can be used in combination with any other diagnostic method useful for the accurate diagnosis of the presence, severity or estimated time of the onset of preeclampsia or eclampsia.

The diagnostic methods described herein can also be used to control and treat preeclampsia in a subject. In one example, if it is determined that a subject has a serum sFlt-1 protein level of 10 ng / ml and a serum free P1GF level of 100 pg / ml, then VEGF can be administered until the level of P1GF in serum reaches approximately 400 pg / ml. In this embodiment, the levels of sFlt-1, P1GF and VEGF or each and every one of these are measured repeatedly as a method not only to diagnose the condition, but also to control the treatment and management of preeclampsia and eclampsia.

Diagnostic Kits

The invention also provides a diagnostic test kit. For example, a diagnostic test kit may include antibodies against sFlt-1, VEGF or P1GF, and means for detecting and more preferably evaluating the binding between the antibodies and the sFlt-1, VEGF or P1GF polypeptide. For detection, the antibody or sFlt-1, VEGF or P1GF polypeptide is labeled, and the antibody or sFlt-1, VEGF or P1GF polypeptide binds to the substrate, such that the interaction of sFlt-1, VEGF polypeptide or P1GF-antibody can be established by determining the

amount of label bound to the substrate after binding between the antibody and the sFlt-1, VEGF or P1GF polypeptide. A conventional ELISA is a common method known in the art for detecting antibody-substrate interaction and can be provided with the kit of the invention. The sFlt-1, VEGF or P1GF polypeptides can be detected in virtually any body fluid including, but not limited to urine, serum, plasma, saliva, amniotic fluid or cerebrospinal fluid. A kit that determines an alteration in the level of sFlt-1, VEGF or P1GF polypeptide with respect to a reference, such as the level present in a normal control, is useful as a diagnostic kit in the methods of the invention.

Selection Essays

As described above, the expression of a sFlt-1 nucleic acid or polypeptide increases in a subject who has preeclampsia, eclampsia or a propensity to develop such conditions. Based on these findings, the compositions of the invention are useful for the selection of low cost and high yield candidate compounds to identify those that modulate the expression of a sFlt-1, VEGF or P1GF nucleic acid molecule whose expression is altered in a subject who has preeclampsia or eclampsia.

Several methods are available to perform screening assays to identify new candidate compounds that alter the expression of a sFlt-1, VEGF or P1GF nucleic acid molecule. In a working example, candidate compounds at varying concentrations are added to the culture medium of cultured cells expressing a nucleic acid sequence of sFlt-1, VEGF or P1GF. The gene expression is then measured, for example, by microarray analysis, Northern blot analysis (Ausubel et al., Supra), or RT-PCR using any appropriate fragment prepared from the nucleic acid molecule as a hybridization probe. The level of gene expression in the presence of the candidate compound is compared with the level measured in a control culture medium lacking the candidate compound. A compound that promotes an alteration such as an increase in the expression of a VEGF or P1GF gene, a nucleic acid or polypeptide molecule, or a reduction in the expression of an sFlt-1 gene, nucleic acid or polypeptide molecule, or a functional equivalent of this is considered useful in the invention; such a molecule can be used, for example, as a therapy to treat preeclampsia or eclampsia in a subject.

In another working example, the effect of candidate compounds at the level of polypeptide production can be measured using the same general strategy and conventional immunological techniques, such as Western blotting or immunoprecipitation with an antibody specific for an sFlt-1, VEGF or P1GF polypeptide. . For example, immunoassays can be used to detect or control the expression of at least one of the polypeptides of the invention in an organism. Polyclonal or monoclonal antibodies (produced as described above), which are capable of binding to such a polypeptide in any conventional immunoassay format (e.g. ELISA, Western blot, RIA assay) can be used to measure the level of the polypeptide. In some embodiments, a compound that promotes an alteration such as an increase in the expression or biological activity of a VEGF or P1GF polypeptide or a reduction in the expression or biological activity of an sFlt-1 polypeptide is considered particularly useful. Again, such a molecule can be used, for example, as therapy to delay, improve or treat preeclampsia or eclampsia, or the symptoms of a preeclampsia or eclampsia in a subject.

In another working example, candidate compounds can be analyzed to detect those that specifically bind an sFlt-1, VEGF or P1GF polypeptide. The effectiveness of such a candidate compound depends on its ability to interact with such a polypeptide or a functional equivalent thereof. Such interaction can be easily tested using any number of conventional binding techniques and functional assays (for example, those described in Ausubel et al., Supra). In one embodiment, a candidate compound can be tested in vitro for its ability to specifically bind to a polypeptide of the invention. In another embodiment, a candidate compound is tested for its ability to reduce the biological activity of an sFlt1 polypeptide by reducing the binding of an sFlt-1 polypeptide and a growth factor, such as VEGF or P1GF.

In another working example, an sFlt-1, VEGF or P1GF nucleic acid is expressed as a transcription or translation fusion with a detectable indicator, and is expressed in an isolated cell (e.g., mammalian or insect cell) under the control of a heterologous promoter, such as an inducible promoter. The cell expressing the fusion protein is then contacted with a candidate compound, and the expression of the detectable indicator in that cell is compared with the expression of the detectable indicator in an untreated control cell. A candidate compound that reduces the expression of a detectable indicator of sFlt-1 or that increases the expression of a detectable indicator of VEGF or P1GF is a compound that is useful for the treatment of preeclampsia or eclampsia. In preferred embodiments, the candidate compound alters the expression of a reporter gene fused to a nucleic acid or a nucleic acid.

In a particular working example, a candidate compound that binds to an sFlt-1 polypeptide can be identified using a chromatography-based technique. For example, a recombinant polypeptide of the invention can be purified by conventional techniques from genetically engineered cells to express the polypeptide (eg, those described above) and can be immobilized on a column. A solution of candidate compounds is then passed through the column and a specific compound is identified for the

sFlt-1 polypeptide based on its ability to bind to the polypeptide and is immobilized in the column. To isolate the compound, the column is washed to remove the non-specific bound molecules, and then the compound of interest is released from the column and collected. Similar methods can be used to isolate a compound bound to a microarray of polypeptides. If desired, the compounds isolated by this method (or any other appropriate method) can be further purified (eg, by high performance liquid chromatography). In addition, these candidate compounds can be tested for their ability to reduce the activity of an sFlt-1 polypeptide or increase the activity of a VEGF signaling pathway (for example as described herein). Compounds isolated by this strategy can also be used, for example, as therapy to treat preeclampsia or eclampsia in a human being. Compounds that are identified as compounds that bind to a polypeptide of the invention with an affinity constant less than or equal to 10 mM are considered particularly useful in the invention. Alternatively, any in vivo protein interaction detection system can be used, for example, any two hybrid assay to identify compounds or proteins that bind to a polypeptide of the invention.

Potential antagonists include organic molecules, peptides, peptide mimetics, polypeptides, nucleic acids and antibodies that bind to a sFlt-1 nucleic acid sequence or an sFlt-1 polypeptide.

SFlt-1 DNA sequences can also be used in the discovery and development of a therapeutic compound for the treatment of preeclampsia and eclampsia. The encoded protein, after expression, can be used as a target for drug selection. In addition, DNA sequences encoding the amino terminal region of the encoded protein or Shine-Dalgarno or other sequences that facilitate translation of the respective mRNA can be used to construct sequences that reduce the expression of a sFlt-1 coding sequence. Such sequences can be isolated by conventional techniques (Ausubel et al., Supra).

Optionally, the compounds identified in any of the assays described above can be confirmed as useful compounds in an assay of compounds that reduce the biological activity of sFlt-1 or that increase the activity of a VEGF signaling pathway.

The small molecules of the invention preferably have a molecular weight of less than 2,000 Daltons, more preferably between 300 and 1,000 Daltons and even more preferably between 400 and 700 Daltons. It is preferred that these small molecules be organic molecules.

Therapeutic Agents Addressing the VEGF Signaling Route

VEGF is a potent mitogen specific for endothelial cells that stimulates angiogenesis, vascular hyperpermeability and vasodilation. Three tyrosine kinase signaling receptors for VEGF have been identified. VEGF receptor binding induces a signaling cascade that results in tyrosine phosphorylation of Cy1 phospholipase, resulting in increases in intracellular levels of 1,4,5-triphosphate inositol and increases in intracellular calcium that activate nitric oxide synthase to produce nitric oxide (NO). The formation of NO activates guanylate cyclase within vascular smooth muscle cells and endothelial cells, causing the production of cGMP. It is believed that this cascade of NO / cGMP mediates the vasoactive effects of VEGF. Another route that seems to be involved in mediating the vasoactive effects of VEGF is the prostacyclin release route. VEGF induces the production of PGI2 through the activation of phospholipase A2 as a result of the onset of the MAPK cascade.

The higher levels of VEGF are useful for the treatment of preeclampsia and eclampsia. Therapeutic compounds that target the VEGF signaling pathways, or the components of a VEGF signaling pathway, and potency in the activity of a VEGF signaling pathway are also useful for the treatment of preeclampsia and eclampsia. Such compounds include sildenafil, prostacyclin analogs, such as Flolan, Remodulin and Tracleer.

Test Compounds and Extracts

In general, compounds capable of reducing the activity of an sFlt-1 polypeptide or increasing the activity of VEGF or P1GF are identified from large libraries of natural products or synthetic (or semi-synthetic) extracts or chemical libraries or of polypeptides or nucleic acids, according to methods known in the art. Specialists in the field of drug discovery and development will understand that the precise source of extracts or test compounds is not critical to the method or methods of selection of the invention. The compounds used in the investigations may include known compounds (for example, known therapeutic agents used for other diseases or disorders). Alternatively, virtually any number of unknown chemical extracts or compounds can be investigated using the methods described herein. Examples of such extracts or compounds include, but are not limited to, plant, fungal, prokaryotic or animal extracts, fermentation broths and synthetic compounds, as well as modifications of existing compounds. Numerous methods are also available to generate random or directed synthesis (eg, semisynthesis or total synthesis) of any number of chemical compounds, including but not limited to compounds based on saccharides, lipids, peptides and nucleic acids. In the market

Synthetic compound libraries are available at Brandon Associates (Merrimack, New Hampshire, United States) and Aldrich Chemical (Milkwaukee, Wisconsin, United States). Alternatively, libraries of natural compounds in the form of bacteria, fungi, plants and animal extracts from various sources are available, including Biotics (Sussex, United Kingdom); Xenova (Slough, United Kingdom), Harbor Branch Oceangraphics Institute (Ft. Pierce, Florida, United States), and PharmaMar (Cambridge, Massachusetts, United States). In addition, natural and synthetic libraries are produced, if desired, according to methods known in the art, for example, by conventional extraction and fractionation methods. In addition, if desired, any library or compound is easily modified using conventional chemical, physical or biochemical methods.

In addition, specialists in the field of drug discovery and development will readily understand that methods of dereplication (e.g. taxonomic dereplication, biological dereplication and chemical dereplication or any combination of these) should be employed whenever possible, or the elimination of replicates or repetitions. of materials already known for their change alteration activity.

When it is discovered that a crude extract reduces the activity of an sFlt-1 polypeptide, or binds to an sFlt-1 polypeptide, additional fractionation of the positive leader extract is needed to isolate chemical constituents responsible for the observed effect. Thus, the objective of the extraction, fractionation and purification process is the careful characterization and identification of a chemical entity within the crude extract that reduces the activity of an sFlt-1 polypeptide. Methods of fractionation and purification of such heterogeneous extracts are known in the art. If desired, compounds that have been shown to be useful as therapeutic agents for the treatment of a human preeclampsia or eclampsia are chemically modified according to methods known in the art.

Therapeutic Agents

The present invention characterizes methods for treating or preventing preeclampsia or eclampsia in a subject. Preferably, the therapeutic agent is administered during pregnancy for treatment or prevention of preeclampsia or eclampsia or after pregnancy to treat preeclampsia or eclampsia after delivery. The techniques and dosages for administration vary depending on the type of compound (antibody, antisense and nucleic acid vector, etc.) and are well known to those skilled in the art or are readily determined.

The therapeutic compounds of the present invention can be administered with a pharmaceutically acceptable diluent, carrier or excipient in unit dosage form. Administration can be parenteral, intravenous, subcutaneous, oral or local by direct injection into the amniotic fluid. The preferred method of administering the therapeutic compounds of the present invention is intravenous administration by continuous infusion.

The composition may be in the form of a pill, tablet, capsule, liquid or sustained release tablet for oral administration; or a liquid for intravenous, subcutaneous or parenteral administration; or a polymer or other sustained release vehicle for local administration.

Well-known methods are found in the art for manufacturing formulations, for example in "Remington: The Science and Practice of Pharmacy" (20th edition, AR Gennaro, editor, 2000, Lippincott Williams & Wilkins, Philadelphia, Pennsylvania, United States) . Formulations for parenteral administration may contain, for example, excipients, sterile water, saline solution, polyalkylene glycols such as polyethylene glycol, vegetable oil or hydrogenated naphthalenes. Biocompatible and biodegradable polymers of lactide, lactide / glycolide copolymer, or polyoxyethylene polyoxypropylene copolymers can be used to control the release of the compounds. Formulations of nanoparticles (eg, biodegradable nanoparticles, solid lipid nanoparticles, liposomes) can be used to control the biodistribution of the compounds. Other potentially useful parenteral release systems include ethylene-vinyl acetate copolymer particles, osmotic pumps, implantable infusion system and liposomes. The concentration of the compound in the formulation varies depending on several factors including the dosage of the drug to be administered and the route of administration.

Optionally, the compound can be administered as pharmaceutically acceptable salts, such as non-toxic acid addition salts or metal complexes that are commonly used in the pharmaceutical industry. Examples of acid addition salts include organic acids such as acetic, lactic, pamoic, maleic, citric, malic, ascorbic, succinic, benzoic, palmitic, suberic, salicylic, tartaric, methanesulfonic, toluenesulfonic or trifluoroacetic acid or the like; polymeric acids such as tannic acid, carboxymethyl cellulose or the like; and inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid or the like. Metal complexes include zinc, iron and the like.

Formulations for oral use include tablets containing the active ingredient (s) in a mixture with non-toxic and pharmaceutically acceptable excipients. These excipients may be, for example, inert diluent.

or fillers (for example sucrose and sorbitol), lubricating, sliding and non-stick agents (for example stearate

of magnesium, zinc stearate, stearic acid, silicas, hydrogenated vegetable oils or talc).

Formulations for oral use can also be provided as chewable tablets, or as hard gelatin capsules in which the active ingredient is mixed with an inert solid diluent or as soft gelatin capsules where the active ingredient is mixed with water or an oily medium.

The dosage and timing of administration of the compound depend on several clinical factors that include the general health of the subject and the severity of preeclampsia symptoms. In general, once preeclampsia or a propensity to develop preeclampsia is detected, a continuous infusion of the purified protein is used to treat or prevent further progression of the condition. The treatment can be continued for a period of time ranging from 1 to 100 days, more preferably from 1 to 60 days and even more preferably from 1 to 20 days, or until pregnancy ends. The dosages vary depending on each compound and the severity of the condition and are titrated to achieve a steady state blood serum concentration ranging from 1 to 500 ng / ml of VEGF or P1GF, or both, preferably 1 to 100 ng / ml, more preferably from 5 to 50 ng / ml and even more preferably from 5 to 10 ng / ml of VEGF or P1GF or both.

Methods to Increase VEGF or P1GF Protein Expression

The present invention characterizes methods to increase the levels of VEGF and P1GF in a subject who has been diagnosed with preeclampsia or eclampsia. Higher levels of VEGF or P1GF can be achieved using several different methodologies described below, among others.

Purified Proteins

In a preferred embodiment of the present invention, purified forms of VEGF or P1GF or both are administered to the subject to treat or prevent preeclampsia or eclampsia.

Purified VEGF or VEGF type proteins include any protein with an amino acid sequence that is homologous, more desirably, substantially identical to the amino acid sequence of VEGF, or any member of the VEGF family, that can induce angiogenesis or that is able to promote the selective growth of vascular endothelial cells or endothelial cells of the umbilical vein. An example of a purified VEGF compound is human recombinant VEGF from Genentech, Inc. (San Francisco, California, United States).

Purified P1GF or P1GF type proteins include any protein with an amino acid sequence that is homologous, more desirably substantially identical to the amino acid sequence of P1GF, or any member of the P1GF family, that can induce angiogenesis or that is capable of promote the selective growth of vascular endothelial cells or umbilical vein endothelial cells. An example of commercially available purified P1GF is human recombinant P1GF from R&D Systems (catalog number 264-PG, R&D Systems, Minneapolis, Minnesota, United States). TromboGenics Ltd is also developing a purified form of P1GF for the treatment of ischemic stroke; Supposedly this form of P1GF would be effective for the applications described in the present invention.

Therapeutic Compounds that Increase the Activity of VEGF or P1GF

The present invention provides the use of any compound known to stimulate or increase blood serum levels of VEGF or P1GF, or the biological activity of these polypeptides, for the treatment or prevention of preeclampsia in a subject. These compounds can be used alone or in combination with the purified proteins described above or any of the other methods used to increase the levels of VEGF or P1GF protein described herein.

An example of a compound that has been shown to stimulate the production of VEGF is nicotine. Although smoking has many risks to the overall health of a pregnant woman and her developing fetus, it is believed that nicotine itself is safer than cigarettes and can be used for short-term therapy in high-risk subjects. Examples include Nicorette (nicotine polacrilex), which is a non-prescription nicotine gum product manufactured by SmithKline Beecham and NicoDerm CQ, which is a nicotine patch sold without a prescription manufactured by Hoechst Marion Roussel Inc. (formerly Marion Merrell Dow). The nicotine released by tobacco is specifically excluded from the methods of the invention in which the patient has not been diagnosed using the methods of the invention.

Nicotine is given after the diagnosis of preeclampsia or eclampsia using the patch or gum. Dosages vary depending on the severity of the condition and the general health of the subject. In general, the manufacturer's instructions are followed to achieve a serum nicotine level ranging from 5 to 500 ng / ml, more preferably from 5 to 100 ng / ml, and even more preferably from 50 to 100 ng / ml.

Theophylline is another example of an additional compound that can be used to treat or prevent preeclampsia or eclampsia. Theophylline is a bronchodilator that is often used for the treatment of asthma and is available

with many trade names (for example, Aerolate Sr, Asmalix, Elxophyllin, etc.) as well as the generic. The administration methods and dosages will vary with each manufacturer and are chosen based on the general health of the subject and the severity of the condition. In general, daily doses vary from 1 to 500 mg, more preferably from 100 to 400 mg and even more preferably from 250 to 350 mg administered twice daily to achieve a serum theophylline level of 5 to 50 μg / ml.

Adenosine is another example of an additional compound that can be used to treat or prevent preeclampsia or eclampsia. Adenosine (Fujisawa Pharmaceutical Co.) is commonly used as an antihypertensive drug. The methods of administration and dosages vary with each manufacturer and are chosen based on the general health of the subject and the severity of the condition. In general, a daily dose of 50 mg / kg administered twice daily is typical for adenosine.

Nifedipine is another example of an additional compound that can be used to treat or prevent preeclampsia or eclampsia. Nifedipine (Bayer Pharmaceuticals) is commonly used as an antihypertensive drug. The methods of administration and dosages vary with each manufacturer and are chosen based on the general health of the subject and the severity of the condition. In general, a daily dosage of 1-2 mg / kg administered twice daily orally or subcutaneously is typical for nifedipine.

Minoxidil is another example of an additional compound that can be used to treat or prevent preeclampsia or eclampsia. Minoxidil (Pfizer, Inc.) is commonly used as an antihypertensive drug. The methods of administration and dosages vary with each manufacturer and are chosen based on the general health of the subject and the severity of the condition. In general, a daily dosage of 0.25 to 1.0 mg / kg administered twice daily orally or subcutaneously is typical for minoxidil.

Magnesium sulfate is another example of an additional compound that can be used to treat or prevent preeclampsia or eclampsia. Magnesium sulfate is a generic drug that is typically used as an antihypertensive drug. The methods of administration and dosages vary with each manufacturer and are chosen based on the general health of the subject and the severity of the condition. In general, a daily dosage of 1-2 g administered intravenously every four hours is a typical dosage for magnesium sulfate.

In addition to the use of compounds that can increase serum levels of VEGF or P1GF, the invention provides the use of any medication for chronic hypertension used in combination with any of the compounds targeting VEGF or P1GF. Medications used to treat hypertension during pregnancy include methyldopa, hydralazine hydrochloride or labetalol. For each of these medications, the modes of administration and dosages are determined by the physician and by the manufacturer's instructions.

Therapeutic Nucleic Acids

Recent work has shown that the supply of nucleic acid (DNA or RNA) capable of expressing an endothelial cell mitogen such as VEGF at the site of a blood vessel lesion will induce proliferation and re-endothelization of the injured vessel. Although the present invention does not refer to blood vessel lesions, in the present invention techniques can also be employed to deliver nucleic acids encoding mitogens of endothelial cells such as VEGF and P1GF used in these studies. These techniques are described in US Pat. UU. 5,830,879 and 6,258,787 and are incorporated herein by reference.

In the present invention the nucleic acid can be any nucleic acid (DNA or RNA) including genomic DNA, cDNA and mRNA, which encodes VEGF or P1GF or any member of the VEGF or P1GF family. The nucleic acid can also include any nucleic acid encoding a protein that has been shown to bind to the sFlt-1 receptor. Nucleic acids encoding the desired protein can be obtained using routine procedures in the art, for example recombinant DNA or PCR amplification.

Therapeutic Nucleic Acids That Inhibit the Expression of sFlt-1

The present invention also relates to the use of antisense nucleobase oligomers to negatively regulate the expression of sFlt-1 mRNA directly. By binding to the complementary nucleic acid sequence (the sense or coding chain), the antisense nucleobase oligomers can supposedly inhibit protein expression by means of enzymatic cleavage of the RNA chain by RNAse H. Preferably , the antisense nucleobase oligomer can reduce the expression of the sFlt1 protein in a cell that expresses excess levels of sFlt-1. Preferably, the reduction in the expression of the sFlt-1 protein is at least 10% with respect to the cells treated with a control oligonucleotide, more preferably 25% and even more preferably 50% or greater. Methods for selecting and preparing oligomers of antisense nucleobases are well known in the art. As an example of the use of antisense nucleobase oligomers to negatively regulate VEGF expression, see US Pat. UU. 6,410,322, incorporated herein by reference. Methods for evaluating protein expression levels are also well known in the art and include Western blotting, immunoprecipitation and ELISA.

The present invention also relates to the use of RNA interference (RNAi) to inhibit the expression of sFlt-1. RNA interference (RNAi) is a recently discovered mechanism of posttranscriptional gene silencing (PTGS) in which a double stranded RNA (mRNA) that corresponds to a gene or mRNA of interest is introduced into an organism resulting in degradation of the corresponding mRNA. . In the RNAi reaction, both the sense chain and the antisense chain of a dsRNA molecule are processed into small RNA fragments or segments that vary in length from 21 to 23 nucleotides (nt) and that have 3 'tails of two nucleotides . Alternatively, synthetic siRNAs that are 21 to 23 nt in length and have 3 'tails of 2 nucleotides can be synthesized, purified and used in the reaction. These 21 to 23 nt rRNAs are known as "guide RNA" or "short interfering RNA" (siRNA).

Then, the siRNA duplexes bind to a nuclease complex composed of proteins that direct and destroy the endogenous mRNA that has homology with siRNA within the complex. Although the identity of the proteins within the complex remains unclear, the function of the complex is to target the homologous mRNA molecule through base pairing interactions between one of the chains of the siRNA and the endogenous mRNA. The mRNA is then cleaved at a distance of approximately 12 nt from the 3 'end of the siRNA and degraded. In this way, specific genes can be directed and degraded, thereby achieving a loss of protein expression of the target gene.

PCT Publication No. WO 01/75164 describes the specific requirements and modifications of cRNA. Although the dsRNA molecules may vary in length, it is most preferable to use siRNA molecules that are mRNAs of 21 to 23 nucleotides with 3 'protruding ends of 2 to 3 nucleotides typically (2'-deoxy) thymidine or uracil. The siRNAs typically comprise a 3 'hydroxyl group. Single stranded siRNAs can also be used as well as blunt end forms of cDNA. In addition, to further improve RNA stability, the 3 'protrusions can be stabilized against degradation. In one of these embodiments, the RNA is stabilized including purine nucleotides, such as adenosine or guanosine. Alternatively, the replacement of pyrimidine nucleotides with modified analogues is tolerated, for example, the replacement of 2-uridine nucleotide projections with (2'-deoxy) thymidine and does not affect the effectiveness of RNAi. The absence of a 2 'hydroxyl group significantly improves the nuclease resistance of the projection in a tissue culture medium.

Alternatively, RNAic can be prepared using any of the methods indicated in the PCT publication

WO 01/75164 or using conventional procedures for in vitro transcription of RNA and tempering procedures of dsRNA as described in Elbashir et al. (Genes & Dev., 15: 188-200, 2001). ARNic is also obtained as described in Elbashir et al. By incubation of dsRNA corresponding to a sequence of the target gene in a Drosophila lysate without cells from Drosophila embryos of syncytial blastoderm under conditions where cRNA is processed to generate siRNA from about 21 to about 23 nucleotides, which are then isolated using techniques known to those skilled in the art. For example, gel electrophoresis can be used to separate the 21-23 nt RNAs and the RNAs can then be eluted from the gel cuts. In addition, chromatography (eg, size exclusion chromatography), glycerol gradient centrifugation and affinity purification with antibodies can be used to isolate RNAs from 21 to 23 nt.

In the present invention, the mRNA or siRNA is complementary to the mRNA sequence of sFlt-1 mRNA and can reduce or inhibit the expression of sFlt-1. Preferably, the reduction in the expression of the sFlt-1 protein is at least 10% with respect to the cells treated with control dsRNA or siRNA, more preferably 25% and even more preferably at least 50%. Methods for evaluating protein expression levels are also well known in the art and include Western blotting, immunoprecipitation and ELISA.

In the present invention, the nucleic acids used include any modification that improves the stability or function of the nucleic acid in any way. Examples include modifications to the phosphate skeleton, the internucleotide bond or the sugar moiety.

To simplify the manipulation and handling of the nucleic acid encoding the sFlt-1 binding protein, the nucleic acid is preferably inserted into a cassette where it is operably linked to a promoter. The promoter must be able to direct the expression of the sFlt-1 binding protein in the desired target host cell. The selection of appropriate promoters can be easily performed. Preferably, a high expression promoter would be used. An example of a suitable promoter is the 763 base pair cytomegalovirus (CMV) promoter. The Rous sarcoma virus (RSV) promoter (Davis, et al., Hum. Gene Ther.

4: 151-159, 1993) and the mouse mammary tumor virus (MMTV) promoter. Certain proteins can be expressed using their native promoter. Other elements that can increase expression (eg, enhancers or a system that results in high levels of expression such as a tat gene and a tar element) can also be included. The recombinant vector may be a plasmid vector such as pUC118, pBR322 or other known plasmid vectors that include, for example, an E. coli origin of replication (see Sambrook, et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press , 1989). The plasmid vector may also include a selective marker such as the �-lactamase gene for ampicillin resistance, provided that the polypeptide marker does not adversely affect the metabolism of the organism that is leaving

to treat The cassette can also be attached to a nucleic acid binding moiety in a synthetic delivery system, such as the system described in PCT Publication No. WO 95/22618.

The nucleic acid can be introduced into the cells by any means appropriate for the vector used. Many of these methods are well known in the field (Sambrook et al., Supra, and Watson et al., "Recombinant DNA", Chapter 12, 2nd edition, Scientific American Books, 1992). Recombinant vectors can be transferred by methods such as calcium phosphate precipitation, electroporation, liposome-mediated transfection, gene gun, microinjection, viral capsid mediated transfer, polybrene-mediated transfer or protoblast fusion. For a review of the procedures for the preparation of liposomes, direction and content delivery, see Mannino and Gould-Fogerite, (Bio Techniques, 6: 682-690, 1988), Felgner and Holm (Bethesda Res. Lab. Focus, 11: 21, 1989) and Maurer (Bethesda Res. Lab. Focus, 11:25, 1989).

The transfer of the recombinant vector (the plasmid vector or viral vectors) can be performed by direct injection into the amniotic fluid or intravenous delivery.

Gene delivery can also be used using adenoviral vectors or adeno-associated vectors (AAV). Adenoviruses are present in a large number of animal species, are not very pathogenic and can replicate equally well in dividing and quiescent cells. As a general rule, adenoviruses used for gene delivery lack one or more genes required for viral replication. Recombinant adenoviral vectors with replication defects used for the delivery of VEGF, P1GF or any sFlt-1 binding protein can be produced according to methods known in the art (see Quantin et al., Proc. Natl. Acad. Sci. States United, 89: 2581-2584, 1992; Stratford-Perricadet et al., J Clin. Invest., 90: 626630, 1992; and Rosenfeld et al., Cell, 68: 143-155, 1992). As an example of the use of in utero gene therapy see US Pat. UU. 6,399,585.

Several methods are available for transfection or introduction of dsRNA or oligonucleotides into mammalian cells. For example, there are several transfection reagents available in the market including, but not limited to: TransIT-TKO ™ (Mirus, Cat. No. MIR 2150), Transmessenger ™ (Qiagen, Cat. No. 301525), and Oligofectamine ™ (Invitrogen, Cat. No. MIR12252-011). Protocols are available for each transfection reagent at the manufacturer.

Once transferred, the nucleic acid is expressed by the cells at the site of the lesion for a period of time sufficient to increase blood serum levels of VEGF, P1GF or any other sFlt-1 binding protein. Since vectors containing the nucleic acid are not normally incorporated into the genome of the cells, the expression of the protein of interest takes place only for a limited period of time. Typically, the protein is expressed at therapeutic levels for about two days to several weeks, preferably for about one to two weeks. A reapplication of DNA can be used to provide additional periods of therapeutic protein expression. Recent examples of gene therapy using VEGF for the treatment of vascular disease in mammals can be found in Deodato et al. (Gene Ther., 9: 777-785, 2002); Isner et al. (Human Gene Ther., 12: 1593-1594, 2001); Lai et al. (Gene Ther., 9: 804-813, 2002); and reviewed in Freedman and Isner (Ann. Intern. Med., 136: 54-71, 2002) and Isner JM (Nature, 415: 234-239, 2002).

Essays for the Expression of Genes and Proteins

The following methods can be used to evaluate the expression of proteins or genes and determine the efficacy for any of the methods mentioned above to increase levels of VEGF, P1GF or any other sFlt-1 binding protein, or to reduce levels of sFlt-1 protein.

The levels of VEGF, P1GF or any protein ligand known to bind sFlt-1 in the subject's blood serum are measured. Methods used to measure serum protein levels include ELISA, Western blotting or immunoassays that use specific antibodies. In addition, in vitro angiogenesis assays can be performed to determine if the subject's blood has been converted from an antiangiogenic state to a proangiogenic state. Such assays are described above in Example 2. A positive result is considered an increase of at least 20%, preferably 30%, more preferably at least 50% and even more preferably at least 60% in serum levels. of VEGF, P1GF or any protein ligand that is known to bind sFlt-1. A positive result can also be considered the conversion from an antiangiogenic state to a proangiogenic state using the in vitro angiogenesis assay.

There are several methods known in the art to evaluate gene expression. Some examples include the preparation of RNA from the blood samples of a subject and the use of RNA for Northern blotting, PCR-based amplification or RNAse protection assays.

Use of Antibodies for Therapeutic Treatment

Elevated levels of sFlt-1 found in serum samples taken from pregnant women suffering from preeclampsia suggest that sFlt-1 is acting as a "physiological fossa" to bind to and reduce trophoblast cells and maternal endothelial cells of VEGF and P1GF functional. The use of compounds, such as antibodies, to bind sFlt-1 and block the binding of VEGF or P1GF can help prevent or treat preeclampsia or eclampsia, resulting in an increase in levels of free VEGF or P1GF. Such an increase would allow an increase in the proliferation of trophoblasts, migration and angiogenesis required for placental development and fetal nutrition, and for the health of systemic maternal endothelial cells.

The present invention provides antibodies that specifically bind to the sFlt ligand binding domain.

1. Antibodies are used to inhibit sFlt-1 and it is believed that the most effective mechanism is by direct blocking of the binding sites for VEGF or P1GF, however, other mechanisms cannot be ruled out. Methods for the preparation and use of antibodies for therapeutic purposes are described in various patents including US Pat. UU. 6,054,297; 5,821,337; 6,365,157; and 6,165,464. The antibodies can be polyclonal or monoclonal; monoclonal antibodies are preferred.

Monoclonal antibodies, particularly those derived from rodents including mice, have been used for the treatment of various diseases; however, there are limitations in its use including the induction of human anti-mouse immunoglobulin responses that results in rapid elimination and a reduction in treatment efficacy. For example, an important limitation in the clinical use of rodent monoclonal antibodies is an antiglobulin response during therapy (Miller et al., Blood, 62: 988-995, 1983; Schroff et al., Cancer Res., 45: 879-885 , 1985).

The matter has attempted to solve this problem by constructing "chimeric" antibodies in which a variable domain binding to an animal antigen is coupled to a human constant domain (US Patent 4,816,567; Morrison et al., Proc. Natl. Acad. Sci. United States, 81: 6851-6855, 1984; Boulianne et al., Nature 312: 643-646, 1984; Neuberger et al., Nature, 314: 268-270, 1985). The production and use of such chimeric antibodies is described below.

Competitive inhibition of the binding of a ligand to sFlt-1 is useful for the prevention or treatment of preeclampsia or eclampsia. Antibodies directed to sFlt-1 can block the binding of VEGF or P1GF to sFlt-1 resulting in higher levels of VEGF or P1GF. Such an increase may result in a rescue of endothelial dysfunction and a shift in the balance of proangiogenic / antiangiogenic factors towards angiogenesis.

A mixture of the monoclonal antibodies of the present invention can be used as an effective treatment for preeclampsia or eclampsia. The mixture may include only 2, 3 or 4 different antibodies or as many as 6, 8 or 10 different antibodies. In addition, the antibodies of the present invention may be combined with an antihypertensive drug (e.g., methyldopa, hydralazine hydrochloride or labetalol) or any other medication used to treat preeclampsia, eclampsia or the symptoms associated with preeclampsia or eclampsia.

Antibody Preparation

Monoclonal antibodies that specifically bind to the sFlt-1 receptor can be produced by methods known in the art. These methods include the immune method described by Kohler and Milstein (Nature, 256: 495-497, 1975) and Campbell ("Monoclonal Antibody Technology, The Production and Characterization of Rodent and Human Hybridomas" in Burdon et al., Editors, Laboratory Techniques in Biochemistry and Molecular Biology, volume 13, Elsevier Science Publishers, Amsterdam, 1985), as well as by the recombinant DNA method described by Huse et al. (Science, 246, 1275-1281, 1989).

Monoclonal antibodies can be prepared from supernatants of cultured hybridoma cells or from ascites fluid induced by intraperitoneal inoculation of hybridoma cells in mice. The hybridoma technique originally described by Kohler and Milstein (Eur. J. Immunol, 6, 511-519, 1976) has been widely applied to produce hybrid cell lines that secrete high levels of monoclonal antibodies against many specific antigens.

The route and immunization program of the host animal or of the antibody producing cells cultured thereof, generally agree with established and conventional techniques for antibody stimulation and production. Normally, mice are used as an assay model; however, any mammalian subject including humans or antibody producing cells thereof, can be manipulated according to the processes of this invention to serve as a basis for the production of hybrid mammalian cell lines, including humans.

After immunization, immune lymphoid cells are fused with myeloma cells to generate a hybrid cell line that can be cultured and subcultured indefinitely, to produce large amounts of monoclonal antibodies. For the purposes of this invention, the immune lymphoid cells selected for fusion are lymphocytes and their normal differentiated progeny, extracted from lymph node tissue or spleen tissue of

immunized animals The use of spleen cells is preferred, since they offer a more concentrated and convenient source of antibody producing cells with respect to the mouse system. Myeloma cells provide the basis for continuous propagation of the fused hybrid. Myeloma cells are tumor cells derived from plasma cells. Murine myeloma cell lines can be obtained, for example, from the American Type Culture Collection (ATCC; Manassas, Virginia, United States). Human myeloma and mouse-human heteromyeloma cell lines have also been described (Kozbor et al., J. Immunol., 133: 3001-3005, 1984; Brodeur et al .; Monoclonal Antibody Production Techniques and Applications, Marcel Dekker, Inc. , New York, pp. 51-63, 1987).

Hybrid cell lines can be maintained in vitro in cell culture medium. Once the hybridoma cell line has been established, it can be maintained in a variety of nutritionally suitable media such as hypoxanthine-aminopterin-thymidine (HAT) medium. In addition, hybrid cell lines can be stored and preserved in any number of conventional ways, including freezing and storage in liquid nitrogen. Frozen cell lines can be revived and cultured indefinitely with a resumed synthesis and secretion of monoclonal antibody. The secreted antibody is recovered from the tissue culture supernatant by conventional methods such as precipitation, ion exchange chromatography, affinity chromatography or the like.

The antibody can be prepared in any mammal, including mice, rats, rabbits, goats and humans. The antibody may be a member of one of the following immunoglobulin classes: IgG, IgM, IgA, IgD or IgE and its subclasses, and preferably is an IgG antibody.

Although the preferred animal for producing monoclonal antibodies is the mouse, the invention is not limited thereto; in fact, human antibodies can be used and may be preferable. Such antibodies can be obtained using human hybridomas (Cole et al., "Monoclonal Antibodies and Cancer Therapy", Alan R. Liss Inc., p 77-96, 1985). In the present invention, techniques developed for the production of chimeric antibodies can be used to help the genes of a mouse antibody molecule of appropriate antigen specificity together with genes of a human antibody molecule (Morrison et al., Proc. Natl. Acad. Sci. 81, 68516855, 1984; Neuberger et al., Nature 312, 604-608, 1984; Takeda et al., Nature 314, 452-454, 1985). Such antibodies are within the scope of this invention and are described below.

As a distinct alternative to the cell fusion technique, immortalized Epstein-Barr virus (EBV) B cells are used to produce the monoclonal antibodies of the present invention (Crawford D. et al., J. of Gen. Virol., 64 : 697-700, 1983; Kozbor and Roder, J. Immunol., 4: 1275-1280, 1981; Kozbor et al., Methods in Enzymology, 121: 120-140, 1986). In general, the procedure is to isolate the Epstein-Barr virus from a suitable source, generally a line of infected cells, and expose the target antibody secreting cells to supernatants containing the virus. The cells are washed and grown in an appropriate cell culture medium. Subsequently, the cells transformed by the virus present in the cell culture can be identified by the presence of the Epstein-Barr virus nuclear antigen and the transformed antibody secreting cells can be identified using conventional methods known in the art. Other methods for producing monoclonal antibodies such as recombinant DNA are also included within the scope of the invention.

Preparation of sFlt-1 Immunogens

The sFlt-1 can itself be used as an immunogen or it can bind to a transport protein or other objects such as sepharose beads. SFlt-1 can be purified from cells known to express endogenous protein such as human umbilical vein endothelial cells (HUVEC; Kendall et al., Biochem. Biophys. Res. Comm., 226: 324-328, 1996) . In addition, nucleic acid molecules encoding sFlt-1 or portions thereof can be inserted into known vectors for expression in host cells using conventional recombinant DNA techniques. Suitable host cells for the expression of sFlt-1 include baculovirus cells (for example, Sf9 cells), bacterial cells (for example, E. coli) and mammalian cells (for example, NIH3T3 cells).

In addition, peptides can be synthesized and used as immunogens. Methods for making antibodies against peptides are well known in the art and generally require coupling the peptide with a suitable transport molecule, such as serum albumin. Peptides include any amino acid sequence that is substantially identical to any part of the sFlt-1 amino acid sequence corresponding to the GenBanK accession number U01134. The peptides can be of any length, preferably 10 amino acids.

or more, more preferably 25 amino acids or more, and even more preferably 40, 50, 60, 70, 80 or 100 amino acids or more. Preferably, the amino acid sequences have an identity of at least 60%, more preferably 85% and even more preferably 95% with the sequence of U01134. Peptides can be obtained commercially or manufactured using techniques well known in the art, such as, for example, the Merrifield solid phase method (Science, 232: 341-347, 1985). The method can use commercially available synthesizers such as the Biosearth 9500 automatic peptide machine, achieving cleavage of the amino acids blocked with hydrogen fluoride, and the peptides being purified by preparative HPLC

using a Waters Delta Prep 3000 instrument, on a 15-20 µm Vydac C4 PrepPAK column.

Functional Equivalents of Antibodies

The invention also includes functional equivalents of the antibodies described herein. Functional equivalents include polypeptides with amino acid sequences substantially identical to the amino acid sequence of the variable or hypervariable regions of the antibodies of the invention. Functional equivalents have binding characteristics comparable to those of the antibodies and include, for example, humanized and single stranded chimerized antibodies as well as their fragments. In the following documents: PCT Publication No. WO 93/21319; European Patent Application No. 239 400; PCT publication

WO 89/09622; European Patent Application No. 338 745; European Patent Application No. 332 424; and U.S. Pat. No. 4,816,567; Methods for producing such functional equivalents are described.

The chimerized antibodies preferably have constant regions derived substantially or exclusively from constant regions of human antibodies and variable regions derived substantially or exclusively from the sequence of the variable region of a mammal other than a human being. Such humanized antibodies are chimeric immunoglobulins, immunoglobulin chains or fragment thereof (such as Fv, Fab, Fab ', F (ab') 2 or other antibody antigen binding sub-sequences) that contain the minimum sequence derived from a non-immunoglobulin. human Methods for humanizing non-human antibodies are well known in the art (as reviews see Vaswani and Hamilton, Ann Allergy Asthma Immunol., 81: 105-119, 1998 and Carter, Nature Reviews Cancer, 1: 118-129, 2001). Generally, a humanized antibody has one or more amino acid residues introduced into it from a source that is not human. These non-human amino acid residues are often referred to as import residues, which are normally taken from a variable import domain. Humanization can be performed essentially following the methods known in the art (Jones et al., Nature, 321: 522-525, 1986; Riechmann et al., Nature, 332: 323-329, 1988; and Verhoeyen et al., Science, 239: 1534-1536, 1988), substituting rodent CDR or other CDR sequences for the corresponding sequences of a human antibody. Accordingly, such humanized antibodies are chimeric antibodies in which substantially less of an intact human variable domain has been replaced by the corresponding sequence of a non-human species (see, for example, U.S. Patent No. 4,816,567 ). In practice, humanized antibodies are normally human antibodies in which some CDR residues and possibly some FR residues are substituted by rodent antibody analog sites (Presta, Curr. Op. Struct. Biol., 2: 593- 596, 1992).

Other methods for the preparation of humanized antibodies can be found in US Pat. Us

 5 821 337 as well as 6 054 297, and Carter, (supra) all of which are incorporated herein by reference. The humanized antibody is selected from any class of immunoglobulins, including IgM, IgG, IgD, IgA and IgE, and any isotope, including IgG1, IgG2, IgG3 and IgG4. When cytotoxic activity is not needed, as in the present invention, the constant domain is preferably of the IgG2 class. The humanized antibody may comprise sequences of more than one class or isotype, and it is within the ordinary skill in the art to select particular constant domains to optimize the desired effector functions.

Human antibodies can also be produced using various techniques known in the art, including phage display libraries (Marks et al., J. Mol. Biol., 222: 581-597, 1991 and Winter et al. Annu. Rev. Immunol., 12 : 433-455, 1994). The techniques of Cole et al. And Boerner et al. Are also useful for the preparation of human monoclonal antibodies (Cole et al., Supra; Boerner et al., J. Immunol., 147: 86-95, 1991).

Suitable mammals other than humans include any mammal from which monoclonal antibodies can be made. Examples of mammals other than humans include, for example, a rabbit, rat, mouse, horse, goat or primate; a mouse is preferred.

Functional antibody equivalents also include single chain antibody fragments, also known as single chain antibodies (scFvs). Single stranded antibody fragments are recombinant polypeptides that normally bind to antigens or receptors; these fragments contain at least one fragment of an antibody variable heavy chain (VH) amino acid sequence chained to at least one fragment of an antibody variable light chain (VL) sequence with or without one or more interconnect linkers. Such a linker may be a short flexible peptide selected to ensure that appropriate three-dimensional folding of the VL and VH domains occurs once they have been associated to maintain the binding specificity of the target antibody molecule of the entire antibody from which the antibody fragment is derived. single chain. Generally, the carboxyl end of the VL or VH sequence is covalently linked by such a peptide linker to the amino acid end of a complementary VL and VH sequence. Single stranded antibody fragments can be generated by molecular cloning, antibody phage display libraries or similar techniques. These proteins can be produced in eukaryotic cells or prokaryotic cells, including bacteria.

Single-chain antibody fragments contain amino acid sequences that have at least one of the variable or CDR regions of the entire antibodies described herein, but that lack some or all of the constant domains of those antibodies. These constant domains are not necessary for antigen binding, but they constitute a major portion of the entire antibody structure. Therefore, single-chain antibody fragments can solve some of the problems associated with the use of antibodies that contain part or all of the constant domain. For example, single stranded antibody fragments tend to be free of unwanted interactions between biological molecules and the heavy chain constant region, or other unwanted biological activity. In addition, single chain antibody fragments are considerably smaller than whole antibodies and, therefore, may have greater capillary permeability than whole antibodies, allowing single chain antibody fragments to be localized and bound to target antigen binding sites. more effectively. In addition, antibody fragments can be produced on a relatively large scale in prokaryotic cells, thereby facilitating their production. In addition, the relatively small size of the single-chain antibody fragments makes it less likely to cause an immune response in a receptor compared to whole antibodies.

Functional equivalents also include antibody fragments that have the same binding characteristics or binding characteristics comparable to those of the entire antibody. Such fragments may contain one or both Fab fragments or the F (ab´) 2 fragment. Preferably, the antibody fragments contain the six CDRs of the entire antibody, although fragments containing less than all such regions such as three, four or five CDRs are also functional.

In addition, the functional equivalents can be or can combine members of any of the following immunoglobulin classes: IgG, IgM, IgA, IgD or IgE, and their subclasses.

Preparation of Functional Equivalents of Antibodies

Antibody equivalents are prepared by methods known in the art. For example, antibody fragments can be prepared enzymatically from whole antibodies. Preferably, antibody equivalents are prepared from DNA encoding such equivalents. DNA encoding antibody fragments can be prepared by removing everything except the desired portion of the DNA encoding the full length antibody.

The DNA encoding chimerized antibodies can be prepared by recombinant DNA that substantially or exclusively encodes human constant regions and DNA that encodes variable regions derived substantially or exclusively from the sequence of the variable region of a mammal other than a human being. DNA encoding humanized antibodies can be prepared by recombination of DNA encoding constant regions and variable regions other than CDRs derived substantially or exclusively from the corresponding human antibody regions and DNA encoding CDRs derived substantially or exclusively from a mammal other than a human being .

Suitable sources of DNA molecules encoding antibody fragments include cells, such as hybridomas, that express the full length antibody. The fragments can themselves be used as antibody equivalents, or they can recombine into equivalents as described above.

The DNA deletions and recombinations described in this section can be performed by known methods, such as those described in the published patent applications indicated above.

Research and Selection of Antibodies

Monoclonal antibodies are isolated and purified using conventional methods known in the art. For example, antibodies can be investigated using conventional methods known in the art such as ELISA against the sFlt-1 peptide antigen or Western blot analysis. Examples of such techniques are described in Examples II and III of US Pat. No. 6 365 157, incorporated herein by reference.

Therapeutic Uses of Antibodies

When used in vivo for the treatment or prevention of preeclampsia or eclampsia, the antibodies of the present invention are administered to the subject in therapeutically effective amounts. Preferably, the antibodies are administered parenterally or intravenously by continuous infusion. The dose and dosage regimen depend on the severity of the disease, and the general state of health of the subject. The amount of antibody administered is typically in the range of about 0.01 to about 10 mg / kg of subject weight.

For parenteral administration, the antibodies are formulated in an injectable unit dosage form (solution, suspension, emulsion) in association with a pharmaceutically acceptable parenteral vehicle. Such

Vehicles are intrinsically non-toxic and non-therapeutic. Examples of such vehicles are water, saline, Ringer's solution, 5% dextrose solution and human serum albumin. Non-aqueous vehicles such as fixed oils and ethyl oleate can also be used. As vehicles, liposomes can also be used. The vehicle may contain minor amounts of additives such as substances that increase isotonicity and chemical stability, for example, buffers and preservatives. Antibodies are typically formulated in such vehicles at concentrations of about 1 mg / ml to 10 mg / ml.

Therapeutic Compounds that Inhibit sFlt-1

Since sFlt-1 levels are elevated in subjects who have preeclampsia, eclampsia or who are prone to develop such conditions, any agent that decreases the expression of a sFlt-1 nucleic acid polypeptide or molecule is useful in the methods of the invention. Such agents include small molecules that can alter the binding of sFlt-1 to VEGF or P1GF, antisense nucleobase oligomers and dsRNA used to mediate RNA interference.

Combination Therapies

Optionally, a therapeutic agent for preeclampsia or eclampsia may be administered in combination with any other conventional therapy for preeclampsia or eclampsia; Such methods are known to the person skilled in the art and are described herein. A therapeutic agent for preeclampsia or eclampsia of the invention can be administered in combination with any compound that increases the activity of a VEGF pathway. Non-limiting examples of agents that also induce the production of endogenous VEGF include nicotine, minoxidil, nifedipine, adenosine, magnesium sulfate and theophylline. In one embodiment, the P1GF protein may be used in combination with any of the agents that induce the production of endogenous VEGF indicated above.

The Control of Subjects

The condition or treatment status of a subject who has preeclampsia or propensity to develop such a condition can be controlled using the methods and compositions of the invention. In one embodiment, the expression of an sFlt-1, VEGF or P1GF polypeptide present in body fluids, such as plasma, is controlled. Such control may be useful, for example, to evaluate the efficacy of a particular drug in a subject or to assess the course of the condition. Therapeutic agents that reduce the expression of a sFlt-1 nucleic acid or polypeptide molecule or that increase the expression of a VEGF or P1GF nucleic acid molecule or molecule are considered particularly useful in the invention.

Claims (14)

one.

A method for diagnosing, in a pregnant person, preeclampsia or the propensity to develop it, said method comprising measuring the level of the sFlt-1 polypeptide in a sample of a subject, wherein said sample is serum or plasma and comparing the level of sFlt- 1 with the level of sFlt-1 in a reference sample where an increase in the level of the sFlt-1 polypeptide in relation to said reference is an indicator diagnosis of preeclampsia or a propensity to develop preeclampsia.

2.

A method for diagnosing preeclampsia in a pregnant person or a propensity to develop it, by measuring the levels of the sFlt-1, VEGF and P1GF polypeptides in a sample of the subject where said sample is serum or plasma, and calculate the relationship between said levels sFlt-1, VEGF or P1GF in a sample of the subject using a metric [sFlt-1 / (P1GF + VEGF)], where an increase in the relationship between these levels in the subject's sample in relation to a reference sample, diagnose preeclampsia or a propensity to develop preeclampsia in that subject.

3.

A method according to claim 1 or 2, wherein a level of sFlt polypeptide greater than 2ng / ml is a diagnostic indicator of preeclampsia.

Four.

The method of claim 1 or 2, wherein said subject is in the first trimester of pregnancy.

5.

The method of claim 1 or 2, wherein said subject is in the second trimester of pregnancy.

6.

The method of claim 1 or 2, wherein said subject is in the beginning of the third trimester of pregnancy.

7.

The method of claim 1 or 2, wherein said measurement is performed using an immunological assay.

8.

The method of claim 7, wherein said immunological assay is an ELISA.

9.

The method of claim 1 or 2, wherein said sFlt-1 is the level of free sFlt-1, linked to total sFlt-1 or sFlt-1.

10.

The method of any one of claims 1, 2, 3, 4, 5 or 6, wherein the sample is serum.

eleven.

A method to diagnose, to a pregnant person, preeclampsia or the propensity to develop it, said method comprising measuring the level of the sFlt-1 nucleic acid molecule in a sample of said subject and comparing it with a reference sample, wherein said The subject's sample is serum or plasma and where an increase in the level of sFlt-1 nucleic acid is a diagnostic indicator of preeclampsia or the propensity to develop preeclampsia.

12.

The method of claim 1, 2 or 11, wherein said measurement of the levels is carried out in two or more occasions and a change in said levels between the measurements is a diagnostic indicator of preeclampsia.

13.

A method for diagnosing, in a pregnant person, preeclampsia or a propensity to develop it, said method comprising measuring the level of free P1GF polypeptide in a serum or plasma sample of said subject where said subject is in the first trimester of pregnancy and in where a statistically significant reduction in free P1GF is an indicator of preeclampsia.

14.

A method for diagnosing, in a pregnant person, preeclampsia, said method comprising measuring the level of free P1GF and creatinine in a urine sample of a subject, where a reduction in the relationship of P1GF with creatinine in the sample in comparison With a control it is a diagnostic indicator of preeclampsia.